The Santa Luiza strain of the southern cattle tick, Boophilus microplus (Canestrini) (Acari: Ixodidae), is resistant to both permethrin and amitraz. A study was conducted at the USDA Cattle Fever Tick Research Laboratory in Texas to investigate the genetic basis of permethrin resistance with cross-mating experiments, and to determine the mechanisms of permethrin resistance through synergist bioassays and biochemical analysis of esterase profiles. The Muñoz strain, an acaricide-susceptible reference strain, was used as the susceptible parent and the Santa Luiza strain, originating in Brazil, was used as the resistant parent. The Food and Agriculture Organization larval packet test was used to measure the levels of susceptibility of larvae of the parental strains, F₁, backcross, F₂, and F₃ generations to permethrin. Results of reciprocal crossing experiments suggested that permethrin resistance was inherited as an incomplete recessive trait. There was no significant maternal effect on larval progeny’s susceptibility to permethrin in the F₁ and subsequent generations. The values of the degree of dominance were estimated at −0.700 and −0.522 for the F₁ larvae with resistant and susceptible female parents, respectively. Results of bioassays on larval progeny of the F₁ backcrossed with the resistant parent strain and of the F₂ generations suggested that one major gene was responsible for permethrin resistance in the Santa Luiza strain. Selection of F₃ larvae with either permethrin or amitraz led to significantly increased resistance to both permethrin and amitraz, indicating a close linkage between genes responsible for permethrin and amitraz resistance. The possible involvement of metabolic enzymes in permethrin resistance in the Santa Luiza strain of B. microplus was dismissed by the lack of enhanced synergism by TPP or PBO, as observed in synergist bioassays, as well as by the lack of enhanced esterase activity in the Santa Luiza strain relative to the susceptible strain. The results of this study suggest that other mechanisms, including a possible new sodium channel mutation that is different from the one currently known, may be responsible for permethrin resistance in the Santa Luiza strain of B. microplus.