Oligonychus punicae and Oligonychus perseae (Acari: Tetranychidae) are the most important mite species affecting avocado orchards in Mexico. Here we used nucleotide sequence data from segments of the nuclear ribosomal internal transcribed spacers (ITS1 and ITS2) and mitochondrial cytochrome oxidase subunit I (COI) genes to assess the phylogenetic relationships between both sympatric mite species and, using only ITS sequence data, examine genetic variation and population structure in both species, to test the hypothesis that, although both species co-occur, their genetic population structures are different in both Michoacan state (main producer) and Mexico state. Phylogenetic analysis showed a clear separation between both species using ITS and COI sequence information. Haplotype network analysis done on 24 samples of O. punicae revealed low genetic diversity with only three haplotypes found but a significant geographical population structure confirmed by analysis of molecular variance (AMOVA) and Kimura-2-parameter (K2P) analyses. In addition, a Mantel test revealed that geographical isolation was a factor responsible for the genetic differentiation. In contrast, analyses of 22 samples of O. perseae revealed high genetic diversity with 15 haplotypes found but no geographical structure confirmed by the AMOVA, K2P and Mantel test analyses. We have suggested that geographical separation is one of the most important factors driving genetic variation, but that it affected each species differently. The role of the ecology of these species on our results, and the importance of our findings in the development of monitoring and control strategies are discussed.