The genomes of all animal species are colonized by endogenous retroviruses (ERVs). Although most ERVs have accumulated defects that render them incapable of replication, fully infectious ERVs have been identified in various mammals. In this study, we isolated a feline infectious ERV (RD-114) in a proportion of live attenuated vaccines for pets. Isolation of RD-114 was made in two independent laboratories using different detection strategies and using vaccines for both cats and dogs commercially available in Japan or the United Kingdom. This study shows that the methods currently employed to screen veterinary vaccines for retroviruses should be reevaluated.