We explored spatial variation in the prevalence of established molecular markers of antimalarial resistance across a geographically diverse, highland region of western Uganda. We identified Plasmodium falciparum CQ resistance transporter 76T mutations in all pools, but there was no evidence of spatial differences across village-based strata defined by either altitude or river valley. In contrast, we identified a significant inverse association between altitude and the prevalence of Plasmodium falciparum multidrug resistance 1 mutations with the largest proportion of Y184F mutations observed in the low-elevation, high-transmission villages. These results demonstrate the substantial heterogeneity in resistance markers observed across geographic settings, even at relatively small scales, but highlight the complex nature of these ecological relationships.