Restriction fragment-length polymorphism analysis of mitochondrial DNA (mtDNA) was used to characterize the genetic variation and population structure of the human bot fly, Dermatobia hominis (Diptera: Cuterebridae), in parasite populations from cattle in southeastern, southern and central regions of Brazil. Forty-eight haplotypes with a nucleotide sequence divergence of 2.75% were found among 227 individuals. Haplotypes could be divided into three clades, with interclade variation ranging from 0.94% to 2.00%. The degree of differentiation obtained suggests that the mitochondrial clades may have differentiated in allopatry, and that their contemporary pattern of distribution probably results from secondary contact of isolated populations, reinforced by the introduction and movement of livestock in South America.