They were previously housed at Port Lympne Zoo in the south of England.
In a statement, the zoo said: “We’re sad to share the news that our two remaining Barbary lionesses at Belfast Zoo, Thheiba and Fidda, have passed peacefully.
“During their lives, the sisters received exceptional care and became key contributors to conservation as part of the EAZA Ex-Situ Programme (EEP), producing multiple offspring and helping secure the future of Barbary lions, a lion sub-species now extinct in the wild.”
They added that in recent weeks Thheiba experienced “mobility issues and weight loss”.
A decision was taken to “euthanise her and end her suffering after a veterinary assessment”.
“Lions are highly social pride animals that rely on companionship within a group.
“Thheiba and Fidda had lived together their entire lives and shared a very strong bond. On veterinary advice, and to avoid significant stress and welfare challenges if left alone, Fidda was also euthanised.”
The zoo said they have had an association with lions for “many decades, with Barbary lions first arriving at the zoo in the 1960s”.
“Thheiba and Fidda’s longevity, their strong bond and their vital role in conservation is testament to our long-standing commitment to the care and conservation of this iconic species.”