Agricultural crop diversity has the potential to alter soil microbial communities and greenhouse gas (GHG) emissions. To test this hypothesis, we conducted a field study on silty clay loam soil in south-east South Dakota under two crop rotations; 2-yr, maize (Zea mays L.)-soybean (Glycine max L.) and 4-yr, maize-soybean-oat (Avena sativa)-winter wheat (Triticum aestivum) managed with a winter cover crop and fallow management under no-till system. Phospholipid fatty acid (PLFAs) profiles were used to assess relative abundance of broad taxonomic groups of soil microorganisms in four active growing seasons. The static chamber technique was used to weekly monitor CO2, CH4, N2O fluxes during the growing seasons of maize and soybean phases in 2017 and 2018, respectively. Total PLFAs and relative abundance of total bacterial and fungal biomass were not affected by the treatments within the sampling event. However, averaged over the study period, the total PLFAs and abundance of total bacterial biomass as well as their sub-groups (Gm+, Gm–, and actinomycetes) were statistically greater with the 4-yr rotation as compared to the 2-yr rotation, whereas, there was no difference between cover crop and fallow plots. Regardless of cover cropping management, the 2-yr rotation had greater CO2 emissions than the 4-yr during the 2017 growing season. However, the 4-yr rotation increased the GHG fluxes during spring thaw of 2018, whereas, its effect was convoluted with cover cropping system (i.e. interaction effect) for summer and fall sampling dates. Cumulative CO2 tended to be greater under cover crop than the fallow when averaged over rotations during 2017 (p = 0.106), however, significant interaction effect during 2018 suggested that cover crops had lower CO2 emissions than the fallow under 2-yr rotation (p = 0.009). This study suggests that cropping system diversification achieved by extending length of rotations through small grains and by growing winter cover crops such as winter rye under no-till system has the potential to alter microbial community composition and mitigate GHG emissions.