Several phytoseiid mite species are important natural enemies used in biological control strategies. In the present study, Cytb mtDNA sequences of various populations of two species, Phytoseiulus macropolis and P. persimilis, were compared to determine whether the specimens collected in Brazil could belong to P. persimilis as this latter species is reported in South America but not in Brazil. The Cytb marker was used because of its high evolution rate, assumed to capture intraspecific variation. No overlap between intra- and interspecific distances was observed but the distances were quite low for interspecific variation. This can be due to the particular biology of Phytoseiulus species and this shows the difficulty to apply a universal threshold in genetic distances to conclude about the existence of one or several species. Cytb mtDNA sequences were also considered to assess intraspecific variation. The DNA sequences of P. persimilis populations were very similar, probably because they all originated from the West Palearctic region or because of a prevalence of commercialized specimens in natura. For P. macropilis, higher genetic distances were observed and differentiation was noted according to geographic location and, to a smaller extent, pyrethroid resistance. To determine how DNA variation might impact the protein function (CytB fragment considered), the amino acid compositions of the populations studied were compared. No diagnostic mutation was observed between pyrethroid resistant and susceptible populations, whereas four mutations were identified between populations of P. macropilis separated by 1300 km (different climatic conditions). The impact of such mutations is discussed but knowledge is scarce, which makes it difficult to root testable hypotheses. The protein analysis clearly opens new perspectives in Phytoseiidae studies.