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In 2019, the World Health Organization (WHO) and the Pan-American Health Organization (PAHO) recognized Mexico as a country free of human rabies transmitted by dogs. Nevertheless, the sylvatic cycle remains as a public health concern in the country. Although cougars (Puma concolor) are not reservoirs of any rabies virus variant (RVV), these felines could act as vectors at the top of the food chain, and their relationships with other organisms must be considered important for the regulatory effect on their prey’s populations. In this study, genetic and antigenic characterization was performed on all cougar rabies cases diagnosed at the Rabies Laboratory Network of the Ministry of Health (RLNMH) in Mexico from 2000 to 2021. Samples from other species, a skunk, a horse (Equus caballus) (attacked by a cougar), and a gray fox (Urocyon cineroargenteus), were included as reference. Rabies cases in cougars were restricted to two Northern states of Mexico (Sonora and Chihuahua). Five out of six samples of cougars were RVV7 (Arizona gray fox RVV) and one from Sonora was RVV1. Interestingly, there is no evidence of RVV1 in dogs in the Northern states since the 1990s but skunk species now harbor this RVV1 in this region of the country.