Surveillance for evidence of West Nile virus (WNV) infection in taxonomically diverse vertebrates was conducted in the Yucatan Peninsula of Mexico in 2003 and 2004. Sera from 144 horses on Cozumel Island, Quintana Roo State, 415 vertebrates (257 birds, 52 mammals, and 106 reptiles) belonging to 61 species from the Merida Zoo, Yucatan State, and 7 farmed crocodiles in Ciudad del Carmen, Campeche State were assayed for antibodies to flaviviruses. Ninety (62%) horses on Cozumel Island had epitope-blocking enzyme-linked immunosorbent assay (ELISA) antibodies to flaviviruses, of which 75 (52%) were seropositive for WNV by plaque reduction neutralization test (PRNT). Blocking ELISA antibodies to flaviviruses also were detected in 13 (3%) animals in the Merida Zoo, including 7 birds and 2 mammals (a jaguar and coyote) seropositive for WNV by PRNT. Six (86%) crocodiles in Campeche State had PRNT-confirmed WNV infections. All animals were healthy at the time of serum collections and none had a history of WNV-like illness.