Rates of nutrient cycling vary across landscape and regional scales. This biogeochemical variability can be partially attributed to patterns in plant community characteristics and abiotic and edaphic conditions across topographic gradients at the landscape-scale or across regional climatic gradients. However, it is also possible that concomitant changes in the microbial communities performing these biogeochemical processes occur across the same spatial scales and may therefore contribute to the observed biogeochemical trends. To assess patterns of microbial community composition across regional and landscape scales, we sampled upland and lowland topographic positions at three grassland communities spanning a 500-mm regional precipitation gradient across the central Great Plains. Soil microbial community composition and biomass were determined using phospholipid fatty acid (PLFA) analysis. Microbial biomass increased across the regional gradient, and different microbial communities were associated with the different grassland community types. The relative abundance of fungi decreased while gram-negative anaerobic bacteria increased from shortgrass steppe to tallgrass prairie. There were no differences in microbial biomass at the landscape-scale, and the only alteration in microbial community composition between upland and lowland landscape positions was a shift toward more nonspecific bacteria in lowlands. The fact that the trends in microbial biomass and community composition at the landscape-scale were less pronounced suggests that variability in microbial community composition is larger regionally across the Great Plains than landscape variability associated with topographical features at any particular site. Alterations in the microbial community may play a role in determining the biogeochemical patterns of grasslands in the Great Plains region.