Australia have been stunned in the final of the women's 4x200m freestyle with China and the USA hunting down the strong favourites and the country's Golden Girls.
Ariarne Titmus confusingly led the race followed by sprint queen Emma McKeon, Madison Wilson and Leah Neal - none of whom featured in the heat.
Neal was yet to swim at all in the Olympics, having only the 4x200m relay on her schedule.
Molly O'Callaghan, just 17-years-old, was unlucky to miss out on the final after posting the 18th fastest time in history in their heat.
Aussie swimming legend Ian Thorpe said he was confused with the team's lineup just before the race as Titmus stepped up to the blocks.
'I'm a little curious about the order they are swimming in' the nine-time Olympic medallist said pre-race, casting justified doubt across Australia.
Australia have been stunned in the final of the women's 4x200m freestyle with China and the USA hunting down the strong favourites and the country's Golden Girls
Ariarne Titmus confusingly led the race followed by sprint queen Emma McKeon, Madison Wilson and Leah Neal - none of whom featured in the heat
Aussie swimming legend Ian Thorpe said he was confused with the team's lineup just before the race as Titmus stepped up to the blocks
Despite Australia leading with their best swimmer China led after the first change.
McKeon took over from the 20-year-old Tasmanian and within the first 50m had overtaken her Chinese opponent, but China still were in the lead at the halfway mark with both teams way ahead of world record time.
Wilson handed over to Neal for the final change still behind China, with Ledecky motoring trying to catch up.
Neal couldn't overcome the Chinese and Americans, leaving the Aussies devastated behind the blocks.
Ledecky posted a lightning fast final 200m to push China all the way to the wall.
The decision to completely overhaul the relay team from heat to final, and lead with their best two swimmers, will be severely scrutinised as it appears Australia have thrown away a gold medal.
Despite Australia leading with their best swimmer, Ariarne Titmus, China led after the first change and all the way until the finish
The decision to completely overhaul the relay team from heat to final, and lead with their best two swimmers, will be severely scrutinised as it appears Aus have thrown away a gold medal
Australia, who were red hot favourites for the event with the individual gold medallist Titmus and sprint queen McKeon in their ranks, were never in control of the race.
The foursome finished with a time of 7:41.29, faster than their heat time of 7:44.61 but the curious decision to select a swimmer who hadn't featured at the Games and start with its fastest two swimmers confused viewers and Thorpe himself.
The Australian swimming legend cast doubt into the audience with his shock at the order of the relay team, and he was justified as China and the USA comfortably held the Aussies at bay.
Titmus, who has been the star of the Games so far for Australia, adds a bronze to her two golds in a lost opportunity to become just the eighth Aussie to win three golds at one Olympics.
She will face-off with Ledecky one more time for the 800m freestyle.
Kyle Chalmers, who touched in an agonising 0.06 seconds behind USA's Caeleb Dressel in the 100m freestyle less than an hour before their relay was supporting from the stands.
Kyle Chalmers, who touched in an agonising 0.06 seconds behind USA's Caeleb Dressel in the 100m freestyle less than an hour before their relay was supporting from the stands
Aussie sprint king Kyle Chalmers has finished second in the final of the 100m freestyle with America's Kyle Dressel taking gold
Earlier the Aussie sprint king touched the wall a finger nail behind Dressel to finish second in the final of the 100m freestyle.
Chalmers, who came from nowhere to win gold as an 18-year-old at the 2016 Rio Olympics, was looking to become the first Aussie to repeat as the fastest man in water.
Chalmers said he was re-watching his 100m freestyle win at the 2016 Olympics this morning in the lead up to the race for inspiration as he attempted to pull off the historic achievement.
The 23-year-old from Port Lincoln touched in behind the USA's world champion by the length of a fingernail for Australia's second silver medal at the Tokyo Games.
'Obviously winning isn't everything… but it's pretty nice,' the devastated swimmer said post race. 'If the games were last year, I wouldn't be here'.
'I'm grateful for the support, my family and friends who have been there since day one. They've always been there for me.'
The Aussies have continued to dominate in the pool with Zac Stubblety-Cook winning gold at the 200m breastroke final on Thursday
Australia's Zac Stubblety-Cook has won gold and broken an Olympic record in the men's 200m breaststroke final at the Tokyo Games
Zac Stubblety-Cook won gold and broke an Olympic record in the men's 200m breaststroke final earlier Thursday.
The 22-year-old from Queensland touched the wall in record time at two minutes and 6.38 seconds followed closely behind the Netherlands' Arno Kamminga and Finnish swimmer Matti Mattsson.
Stubblety-Cook had been a favourite to win after taking out first place in the semis with the fastest time of two minutes and 7.35 seconds.
No Australian had won the 200m breaststroke final at the Olympics since the previous Games in Tokyo in 1964.
Speaking after his sensational swim to Channel 7, Stubblety-Cook said he was 'lost for words' while his family and long-term girlfriend Ella Martinkovic cheered him on from home.
'Unbelievable, without their support I wouldn't be here but it's been a tough five years and I'm lost for words to be honest,' he said.