Nesari died in her sleep of old age May 26, Dvur Kralove zoo spokeswoman Jana Mysliveckova said. She called the death "an irretrievable loss."
The rhino was brought to the zoo in 1975 from Sudan. Nesari's death leaves the zoo with one remaining northern white rhino, 30-year-old Nabire.
In an attempt to save the species from extinction, the Czech zoo moved four of its northern white rhinos to a game park in Kenya in December 2009, hoping to be easier for them to breed there than in captivity.
Mysliveckova said few of these rhinos are now left: two at a zoo in San Diego while three or four who are believed to live in Sudan have not been seen since last year.