The cattle tick, Rhipicephalus annulatus (Say) is a vector of bovine babesiosis and responsible for direct and indirect losses to cattle producing areas located in temperate and subtropical dry regions. Resistance against pyrethroids has been reported for this species in Asia and Africa, but never before in North America. An outbreak strain, Rio Lado, collected close to the border between Mexico and the United States, in Maverick County, Texas, showed low level of resistance to permethrin, a pyrethroid pesticide. We used genomic material from different strains of cattle ticks collected within the Permanent Quarantine Zone (Rio Lado, Vega and Klein Grass strains) to partially characterize the coding gene of the voltage-gated sodium channel (Na-channel), target-site of pyrethroid pesticides, and search for putative mutations associated with resistance using quantitative PCR high resolution melt (HRM) analysis. The two amplified fragments, corresponding to domains II and III of the Na-channel, were 100 % identical to its ortholog in Rhipicephalus microplus (Canestrini). No nucleotide polymorphisms in the Na-channel gene were observed in the pyrethroid-resistant Rio Lado strain, when compared to the susceptible strains Klein Grass and Vega. This study reports the first case of pyrethroid resistance in R. annulatus collected in the United States. Also, we provide new genomic data for this species of tick that allows for the development of a new method to screen for mutations associated with pyrethroid resistance.