This study explores potential signals of microdifferentiation in the gene pool of three high-altitude populations from Jujuy province in northwest Argentina using highly polymorphic markers. These human communities are characterized by extreme living conditions and very low population densities owing to considerable height above sea level and steep orography. A set of autosomal short tandem repeats (STRs) located at chromosome 6 (6p21.3) was typed in samples from Quebrada Baja (∼2,500 m), Quebrada Alta (∼3,300 m), and Puna (> 3,500 m). Genetic diversity was estimated through the observed and expected heterozygosities and the haplotype diversity. Analyses of the molecular variance (AMOVAs) and population differentiation tests based on allele and haplotype frequencies were performed to assess genetic heterogeneity among subgroups. No deviation from Hardy-Weinberg equilibrium was detected in any subpopulation, yet significant departures were detected in the analysis considering the whole area (D6S2792 and D6S105 loci). Overall, genetic diversity showed a decreasing trend as the altitude increased. Thus, allele and haplotype frequencies showed the most significant differences between Puna and Quebrada Baja, the populations sited at the edges of the altitude range. The trend toward reduction of heterozygosity with altitude is compatible with historical patterns of colonization, interregional migration trends, population density, and genetic admixture. The main consequence of the complex mountainous landscape of Jujuy would be an imbalance in the interplay of gene flow and genetic drift, favoring the latter. The combined effect of restricted gene flow and intense genetic drift would have promoted local genetic differentiation between the Jujuy highland subpopulations, leading to spatial patterning of the allele frequencies not entirely attributable to geographic distance. Our findings corroborate the effectiveness of STRs to identify microevolutionary changes.