Chemical control based on the use of pyrethroid and organophosphate compounds has selected resistant genotypes in populations of Rhipicephalus (Boophilus) microplus. Point mutations in esterase-encoding genes represent one of the main resistance mechanisms in this species. In this study, the PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) technique was used to investigate the presence of mutations in a fragment of a putative carboxylesterase in a population of ticks with a history of resistance. The digestion of a fragment of 372 pb with EcoRI revealed three genotypes: W, H and M, observed in different frequencies. The homozygous wild-type genotype (W) was detected only in sensitive strains, with high frequency. The heterozygous genotype (H) was observed in all the strains, albeit with higher frequency in the strains with a moderate resistance, while the homozygous mutant genotype (M) was found only in the moderate resistant strain and resistant strains, with higher frequency in the resistant strains. A comparison of the sequences indicated the presence of other mutations, besides EcoRI polymorphism in the moderate resistant and resistant strains. Also found was the presence of stop codons generating truncated proteins in the sensitive and moderate resistant strains. A domain analysis revealed the presence of additional domains in the resistant strain. These findings suggest that different point mutations, as well as the influence of post-translational modification mechanisms, are altering the activity of the translated proteins and may be associated with resistance.