Single-strand conformation polymorphism (SSCP) analysis was examined in a 303-bp region of the 16S and 12S mitochondrial rDNA genes to study haplotype frequencies among populations of Gulf Coast ticks collected from Refugio Co., TX, Payne Co., OK, and two sites in Osage Co., KS. Seven haplotypes were identified from the 16S rDNA gene fragment, whereas only two haplotypes were detected from the 12S fragment. Only the results from the 16S rDNA fragment are discussed. Haplotype diversity was greatest in Kansas (site 1), where three of the four haplotypes detected were unique to this site. All Gulf Coast tick populations shared the fourth haplotype. Two haplotypes were determined for Texas and Oklahoma populations, one of which appeared only in Texas, whereas the other was shared. Nei's haplotype diversity (h) indicated that the Texas population was relatively homogeneous (15%), whereas the remaining populations were heterogeneous (42–59%), although the Bonferroni confidence interval found no significant differences (P < 0.05). Nucleotide sequencing of the seven haplotypes and subsequent phylogenetic analysis using neighbor joining showed a monophyletic relationship among these haplotypes. One haplotype, shared by both Oklahoma and Kansas (site 2), was basal to the remaining haplotypes and formed a distinct clade. Two haplotypes, both from Kansas (site 1), formed a unique clade, whereas the remaining four haplotypes were unresolved polytomies.