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.

On 5 April 2014 voters went to the polls in a carnival atmosphere, despite heavy rain. But more than two-thirds of cabinet posts are still unfilled.
The decision by President Ghani to suspend all provincial governors and police chiefs has led to the further stagnation of government across the country.
The reformist governor of Nangarhar province in the east has resigned from the post because he was left without the power he needed to do his job.
Nangarhar is in a crucial location, both in terms of security as well as revenue-raising, as it is the gateway to Afghanistan from the Khyber Pass in the east.
Its provincial capital, Jalalabad, has been facing increasing attacks from the Taliban and other insurgents in recent months.
At a public meeting, a number of elders signed a letter urging the governor, Mullah Ata Ullah Ludin, not to stand down. One said that he was the only person taking on the "criminal mafia".
Mr Ludin's departure leaves this important province leaderless. He finally insisted he had to step down because he could not make the decisions he wanted.
His "acting" status meant he was prevented from appointing new teachers, and an order he gave to close a border weighing station was overturned.
He had made the decision because trucks were constantly leaving far heavier than had been weighed - and must have paid a bribe for the difference.
Apart from the lost revenues, the heavy trucks were damaging the region's roads.  He said that if the situation of acting governors and police chiefs was not resolved soon, then the country would "slide towards instability; people will lose faith in the government and corruption will increase".
It's the same story in the province of Herat, the gateway to Iran, at the western end of Afghanistan's most important trade route.
Stagnation here following the suspension from office of most senior officials has provoked the former governor Ismail Khan to actively campaign against President Ghani's government.
Khan was a prominent commander in the jihadi war against the Soviet Union in the 1980s. He and other ex-jihadi commanders say they are locked out of government, and are a formidable opposition to the technocratic moderniser who is now president.
But President Ghani is also losing support among those who previously backed him.
MPs like Helai Ershad say that far from establishing a reformist government, he has had to make alliances with men she described as "a bunch of mujahidin".
She said that the problems began when the international community persuaded Ghani to share power with Abdullah Abdullah, the opponent he narrowly defeated in a contested election.
Since both men have two deputies, there are six separate powerbases to satisfy in making appointments.
"Two different mentalities, how can they work under the same umbrella?," she asked.
Since there are no political parties in Afghanistan, a country where patronage is the main driver of power, the jockeying for position has gone on.
Ms Ershad believes that only 10 of the more than 20 ministers still to be confirmed by parliament will be approved this time round.
And the issue of appointing substantive provincial governors and police chiefs has still to be addressed.
A pair of sloths at an East Sussex zoo failed to breed after it emerged that they were both in fact males.
[NEXT_CONCEPT]
Afghanistan still does not have a full government a year after the presidential election.