The animal mithocondrial genome has been widely used as an efficient molecular marker for population genetic studies in a wide range of insect species. In this work we analyze three specific regions of the horn fly, Haematobia irritans, mtDNA using PCR-RFLP:  Amplicon 1 (2100bp-2700bp), containing the control region; Amplicon 2 (1700bp), containing the genes COXIII, ATPase-6 and ATPase-8; and Amplicon 3 (650bp), containing the tRNAs cluster between NADH5 and NADH3 genes. Preliminary analysis of Amplicon 1 for samples from Argentina, Uruguay, Venezuela and Brazil allowed the detection of size polymorphism (about 600bp) in samples from Venezuela and Brazil, however no polymorphic PCR-RFLP patterns were found. The occurrence of ins-del events in the control region may be responsible for the observed size variation reported in this work. Analysis of Amplicon 2 indicates monomorphic restriction patterns for DraI, EcoRV, PvuII, XbaI and XhoI. Sequencing of Amplicon 3 is being conducted for structural analysis and identification of restriction sites for further PCR-RFLP analysis. The identification of length polymorphism for Amplicon 1 was an uncommon feature which may provide a potential marker for population genetic studies. Increasing numbers of individuals from these populations and additional populations from other geographic regions are being analyzed for a better characterization of H. irritans genetic variability. These results may contribute to understanding horn fly introduction and spread through South America. Financial Support: CNPq/PROFIX and FAPESP.