Cropping systems are structured to maximize crop yields and increase sustainability in agricultural production. A field study was conducted to investigate different long-term cropping systems on soil organic matter and microbial communities. The cropping systems studied were: (i) a 14-year continuous alfalfa (Medicago sativa L.) (CA), (ii) a 9-year alfalfa removed and rotated with 4–5 years continuous annual crops (spring wheat (Triticum aestivum L.), maize (Zea mays L.), potato (Solanum tuberosum L.), and millet (Panicum miliaceum L.)), and (iii) a 5-year field fallow after alfalfa. Results showed that continued annual crops decreased total organic C and labile organic C by 10 to 20% and 17 to 34% in the topsoil (0–30 cm), and by 15 to 35% and 20 to 46% in the subsoil (30–60 cm), respectively, compared with CA. Similar trends were found in soil total N concentration, which decreased by 7 to 20% in the topsoil. Highest microbial biomass C was found in CA. Shannon-Wiener diversity and substrate richness of soil microbes measured by Biolog EcoPlates was significantly affected by cropping system with CA exhibiting a higher degree of soil microbial functional diversity in the topsoil, while the lowest values were found in the alfalfa-potato system. The higher soil organic matter content and functional diversity of soil microbe in CA indicates that soil nutrition and microbial activity did not limit alfalfa development and growth in the dryland area. The lower microbial activity and functional diversity observed in the potato field indicates the importance of crop selection in cropping systems.