Boophilus microplus (Canestrini) (Acari: Ixodidae), collected in Hidalgo County, TX, were determined to be resistant to permethrin. Discriminating dose (DD) tests at the LC₉₉ and 2× the LC₉₉ of susceptible ticks yielded lower than expected mortalities for permethrin but not for coumaphos or amitraz acaricides. Initial bioassay results confirmed the pyrethroid resistance detected in the DD assays. Two generations of selection with permethrin at a rate >60% increased the measured resistance ratios from 9.5 (7.9–11.5) to 263 (217–320). Synergist studies suggested that metabolic enzymes were involved in permethrin resistance. Native gel electrophoresis verified that the CZS9 esterase was not involved in resistance to permethrin. Polymerase chain reaction examination revealed the presence of a mutation of the sodium channel gene (Phe[arrow]Ile amino acid substitution in the S6 trans-membrane segment of domain III), in the resistant B&H population. The frequency of this mutation increased after selection with permethrin and concurrent increase in estimated resistance ratios. The B&H population was eradicated from the United States by the U.S. Department of Agriculture’s Animal and Plant Health Inspection Service Veterinary Services, Cattle Fever Tick Eradication Program through the use of the organophosphate acaricide coumaphos.