In this study, we explore variation in resource use among Streptomyces in prairie soils. Resource use patterns were highly variable among Streptomyces isolates and were significantly related to location, phylogeny, and nitrogen (N) amendment history. Streptomyces populations from soils less than 1 m apart differed significantly in their ability to use resources, indicating that drivers of resource use phenotypes in soil are highly localized. Variation in resource use within Streptomyces genetic groups was significantly associated with the location from which Streptomyces were isolated, suggesting that resource use is adapted to local environments. Streptomyces from soils under long-term N amendment used fewer resources and grew less efficiently than those from non-amended soils, demonstrating that N amendment selects for Streptomyces with more limited catabolic capacities. Finally, resource use among Streptomyces populations was correlated with soil carbon content and Streptomyces population densities. We hypothesize that variation in resource use among Streptomyces reflects adaptation to local resource availability and competitive species interactions in soil and that N amendments alter selection for resource use phenotypes.