Since the discovery of Drosophila pseudoobscura in the tropical highlands of the Colombian Andes during the 1960s, this population has been studied by many evolutionary biologists because of its geographical isolation from the main North American range of this species. We used five highly variable microsatellite loci (DPSX001, DPS2001, DPS3001, DPS3002, and DPS4001) to analyze the genetic structure of three Colombian populations and the genetic relationships with four North American populations. We found that the average heterozygosity was consistent among the three tropical Colombian populations (H = 0.665–0.675), but they had less variability than their North American counterparts. Nonetheless, the genic diversity found in the Colombian populations was higher than that found previously using other genetic markers. The average genic heterogeneity estimate among the Colombian populations (R_ST = 0.042), although statistically significant, was substantially lower than that found among the North American populations (R_ST = 0.088). We identified alleles in the Colombian populations not reported in North American populations, suggesting further divergence between the populations. We estimated that the populations on the two continents diverged ca 80,000 years ago, consistent with independent sequence analyses of these populations but contrary to some suggestions in the literature. Finally, we estimated an average effective population size of the Colombian populations to be on the order of 100,000.