We used mitochondrial DNA (mtDNA) sequence data and allele frequencies at eight microsatellite loci to examine the population genetic structure, estimate the divergence times of distinct lineages, and infer patterns associated with host colonization in populations of the bark beetle Dendroctonus approximatus in Mexico. Two haplotype groups were identified using mtDNA sequences in 71 individuals from 15 populations. The first group was distributed in the Sierra Madre Occidental (SMOc, Western Mexico), with some populations in the Faja Volcánica Transmexicana (Central Mexico), and the second was found in the Sierra Madre Oriental (SMOr, Eastern Mexico), with populations in the Sierra Madre del Sur (Southern Mexico). The estimated split between groups occurred in the late Pleistocene, around 0.195 Mya. Microsatellite allele frequencies revealed high genetic differentiation between pairwise populations, and genetic differentiation values indicated a genetic structure of isolation by distance. Both mtDNA sequence data and microsatellite allele frequencies indicated that D. approximatus had two independent colonization routes in Mexico, one through the SMOc and another along the SMOr. The widespread geographic distribution of D. approximatus in Mexico follows a model of population range expansion of two haplotype groups in which gene flow is restricted by the geographic separation between hosts imposed by physical barriers between populations.