Juntos, a humboldt penguin, died at the Calgary Zoo on Tuesday.
The death comes the day after Guillermo, another humboldt, also died.
The zoo reported on its Facebook page that senior staff veterinarian Dr. Doug Whiteside believes the pair, both three-years-old, died of an infection.
While he is waiting for confirmation from lab tests, the results are most consistent with an infection caused by the bacterium Chlamydophila psittaci.
"Our animal care staff members including veterinary staff, curators and keepers are working feverishly to prevent further losses in our group of eight humboldts and two gentoos that share the outdoor exhibit," read the zoo’s statement.
"This disease has caused outbreaks in other zoos in penguin groups and has been detected in wild humboldts and other bird species. The disease can be latent in birds for months or years so it’s possible the humboldts arrived in Calgary as carriers of Chlamydophila, undetected despite comprehensive pre-shipment and quarantine testing."
Both birds came to the zoo from the Rosamond Gifford Zoo in Syracuse, New York.
"Our humboldts have just completed their annual molt where they naturally shed all their feathers over a period of two to three weeks and during this time of physiological stress the birds can lose as much as 20 per cent of their body weight making them more susceptible to infections," zoo officials said.
The birds will stay in their outdoor exhibit area while they're being treated and will be kept separate from the penguins that live in the indoor exhibit at Penguin Plunge.