Clinically healthy reptiles may shed Salmonella and therefore act as a potential zoonotic threat. Most people in Northern European countries are rarely exposed to reptiles, but many zoos have education departments where children have direct contact with this group of animals. The objectives of this study were to determine the prevalence and serotype distribution of Salmonella among reptiles in the Education Department (n = 55) at Copenhagen Zoo and compare it to the Zoo’s main reptile collection (n = 145) to evaluate the zoonotic risk. Salmonella was isolated from cloacal swabs by selective enrichment, and a single isolate from each positive sample was further identified by biochemical tests and serotyped. The overall prevalence was 35% (69/200) with significant difference between the Education Department (64%, 35/55) and the main reptile collection (23%, 34/145). A total of 28 serotypes were detected. Ten serotypes were isolated from more than one specimen and four from more than one species. Salmonella enterica subsp. enterica serovar Eastbourne was the predominant serotype (32%, 22/69) and was also the serotype isolated from most reptile species (n = 7). Transmission of serotypes from one department to another was very limited indicated by the serotype distribution. Despite the relative high prevalence observed among the reptiles in the Zoo’s Education Department compared to the reptiles in the Zoo’s main reptile collection, no Salmonella cases have been linked to the Zoo, and Salmonella ser. Eastbourne is very rarely isolated from humans in Denmark. Simple hygienic procedures such as hand washing which is consistently carried out following handling of reptiles at the Education Department may reduce the risk and therefore contribute to this low prevalence.