Keepers at Drusillas Park in Alfriston could not work out why Sofia and male Tupee had failed to bond since being introduced to each other in 2011.
Head keeper Mark Kenward said it was not easy to establish the sex of a sloth and medical records had shown Sofia was female when she arrived.
"We are going to have to find a more suitable name for Sofia," he said.
It is likely that either Tupee or Sofia will now be re-homed to another zoo, and another female introduced.
Mr Kenward said: "We had never looked after sloths before and were relying on the information provided.
"Unfortunately we cannot keep our two boys together and it would also be a waste of their breeding potential."
After enlisting the help of Bristol Zoo, Mr Kenward said it all made "perfect sense" given the problems they had been experiencing with the animals.
A pair of sloths at an East Sussex zoo failed to breed after it emerged that they were both in fact males.