It is assumed that geographic isolation of Dendroctonus Erichson species populations or their plant hosts determines genetic structure. This structure can be analyzed with respect to the biogeographic pattern that describes the species in a region. The Trans-Mexican Volcanic Belt (TMVB) is located between the Neartic and Neotropical regions and is a center of diversification and endemism of trees in the genus Pinus L. Dendroctonus mexicanus Hopkins is polyphagous within Pinus species and has a continuous geographic distribution across the TMVB. We explored whether the population genetic structure of D. mexicanus is reflective of the distribution pattern of the Dendroctonus species that occur in the TMVB. Twelve gene loci were analyzed by isozyme electrophoresis in 17 populations found on pines from the Leiophyllae subsection. Allele frequencies, average heterozygosity, heterozygosity by locus, deviations from Hardy–Weinberg equilibrium (HWE), F-statistics among populations, and average genetic flow were calculated. Genetic structure was determined using the relationship between F_ST versus geographic distances among populations. Genetic relations among populations were established by neighbor-joining and principal components analysis by using Nei’s genetic distances. Dendrogram reliability was assessed by bootstrap analysis and cophenetic correlation coefficient by using the Mantel test. Results show that heterozygosity of D. mexicanus is similar to other scolytids. A high proportion of loci were out of HWE by homozygous excess, which may be explained by multiple factors. The scarce number of fixed alleles, the allele variation pattern, pairwise genetic distances, and F-statistics suggest a model of isolation by distance for D. mexicanus in the TMVB resulting from recent dispersal events.