Polymerase chain reaction (PCR) diagnostic assays were used to identify possible resistance-associated roles of two amino acid substitutions found in pyrethroid resistance-associated genes of Boophilus microplus (Canestrini). Individual larvae from the San Felipe target site resistant strain and the Coatzacoalcos (Cz) metabolic resistant strain were separated into resistant and susceptible groups by larval packet bioassays and analyzed by PCR. A Phe → Ile amino acid mutation in the sodium channel gene S6 transmembrane segment of domain III was found to have a close association with survival of acaricide treatments containing as high as 30% permethrin. As the permethrin dose was increased, an increase was seen in the proportion of surviving larvae that possessed two mutated sodium channel alleles. An Asp → Asn amino acid substitution, originally found in high allele frequency in alleles of the CzEst9 esterase of the Cz strain, appeared to provide some resistance to permethrin. However, the presence of the mutation did not associate with resistance in the dose–response fashion seen with the sodium channel amino acid mutation. Resistance provided by CzEst9 might be more dependent on concentration of CzEst9 more so than the presence of a mutated allele.