Five other endangered pups appear to be thriving and are being cared for by their mother Millie, 8, and father Graham, 9, in a den at the Red Wolf Woods exhibit.
The litter – which included four males and four females – was born over a 30-hour period starting on Mother’s Day.
Zoo staff has not determined the genders of the two who died, and said they were the smallest in the litter.
Although red wolf litters can include up to nine pups, the average litter is four, said Will Waddell, the zoo’s red wolf program coordinator.
The pups were the first born on zoo grounds in 29 years.
Zoo biologists have checked on the wolves but are trying to give Millie and Graham space to care for their pups without much interference.