The undulating topography of Prairie Pothole Region of North America creates spatial and temporal variability in soil moisture and nutrient levels, affecting microbial community processes and greenhouse gas emissions. By identifying differences in soil bacterial and archaeal community composition and the abundance of nitrogen cycling genes in permanent cover versus annual crop land over two growing seasons (2017 and 2018), we were able to assess the effects of topography and land use on the functional capacity of the soil microbiome. Permanent grassland cover was associated with higher bacterial diversity in upland positions and lower diversity in low-lying depressions. Bacterial community composition was also significantly different between cultivated and permanent cover at all points along the topographic slope, with the largest effects seen in the footslope and backslope positions. Compared to permanent cover, soil from annual cropland had consistently more abundant nitrifiers, including Nitrospira in the toeslope and backslope, and Nitrososphaeraceae in the shoulder and knoll samples while soils from permanent cover had a greater abundance of several Alphaproteobacteria from Rhodospirillales and Hyphomicrobiaceae across multiple upland positions. Upland soils from annual cropland also had consistently higher abundance of both bacterial and archaeal ammonia oxidizing (amoA) genes and a higher ratio of nirK:nirS genes compared to those from permanent cover. These differences in microbial community composition were associated with higher N₂O and CO₂ emissions in upland soils in annual cropland; however, there were no differences in GHG emissions between the two systems in low-lying positions.