Soil ecosystem properties and processes which simultaneously maintain native fertility and sustain plant yields are of principal interest in sustainable agriculture. Native prairies in Kansas are relevant in this context, as they have been annually hayed with no fertilization or detectable decline in yield or soil fertility. In contrast, intensive wheat production has resulted in significant reductions in soil fertility and now requires intensive inputs to maintain yield. This study aimed to shed light on the soil microbiological differences between these two contrasting agricultural systems in an attempt to gain insight into possible mechanisms driving nutrient and energy efficiencies in these hayed prairie ecosystems. The objectives of this study were: i) to identify major differences in soil bacterial and nitrogen fixing communities between prairies and adjacent annual wheat fields, ii) to determine if dramatic losses of soil organic carbon (SOC) are a result of obsolete farming practices, or from plant community composition, and iii) to document the relative contribution of associative N-fixation to total plant N in three C4 prairie grasses. Soil analyses, microbial biomass, and terminal restriction fragment length polymorphism analyses (T-RFLP) revealed that bacterial and nitrogen fixing communities that were correlated with soil chemical, physical, and biological properties indicative of higher soil quality in prairie sites. In addition, SOC loss was documented in annual agriculture fields, even in the absence of tillage, demonstrating the large role that prairie plant communities play in maintaining soil fertility. Finally, evidence of associative N fixation was found in prairie grasses which may help alleviate N limitations and sustain long-term exports of N. Two additional studies were conducted to advance T-RFLP methodology. The first study was an evaluation of statistical multivariate analyses for T-RFLP data and yielded insight into which analyses were most appropriate given research objectives and dataset complexity. The second study yielded T-REX, a free, online software for rapid and less-biased analyses of T-RFLP data. Collectively, the results of this work suggest a greater synchrony of plant nutrient demand in prairies, which may help to explain the greater nutrient use efficiencies seen in these systems relative to wheat.