The genetic population structure relationships of Hyalomma (Euhyalomma) lusitanicum in Andalusia (the south of the Iberian Peninsula) were examined using mtDNA sequence data from 887 bp of cytochrome oxidase subunit I (COI) gene. The sequence for the COI region was determined for 84 individuals collected in several localities of Andalusia, and 10 for other localities (i.e., five from Toledo, central Iberian Peninsula, four from Sicily (Italy) and one from Canary Island). Seventeen haplotypes were detected, including 27 polymorphic sites. The number of amino acid substitutions per site from mean diversity calculations for the entire population was 0.017. AMOVA analysis revealed a low gene flow that characterises the genetic population structure of this species in South Iberian Peninsula, with a haplotype diversity (h) value of 0.815. No geographically induced differentiation was observed, and separate evolutionary units were not detected. Our results indicate low genetic diversity across the geographical range of H. lusitanicum tick in Andalusia. Our data do not show any genetic discontinuity between the tick populations studied, including specimens from Canary Island and Sicily (Italy).