West Nile virus (WNV) was probably introduced in southern and northern Mexico from the USA in two independent events. Since then, WNV activity has been reported in several Mexican states bordering the USA and the Gulf of Mexico, but disease manifestations seen there in humans and equids are quite different to those observed in the USA. We have analysed WNV seroprevalence in asymptomatic, unvaccinated equids from two Mexican states where no data had been previously recorded. WNV IgG antibodies were detected in 31.6% (91/288) of equine sera from Chiapas and Puebla states (53.3% and 8.0%, respectively). Analysis by plaque reduction neutralization test (PRNT) showed good specificity (99.4%) and sensitivity (84.9%) with the ELISA results. Further analyses to detect antibodies against three different flaviviruses (WNV, St Louis encephalitis virus, Ilheus virus) by haemagglutination inhibition (HI) tests on a subset of 138 samples showed that 53% of the 83 HI-positive samples showed specific reaction to WNV. These data suggest continuous expansion of WNV through Mexico.