AbstractThere is a growing awareness of the importance of soil microorganisms in agricultural management practices. Currently, much less is known about whether different crop cultivar has an effect on the taxonomic structure and diversity, and specific functions of soil bacterial communities. Here, we examined the changes of the diversity and composition and enzyme‐encoding nitrogenase genes in a long‐term field experiment with seven different rhizoma peanut cultivars in southeastern USA, coupling high‐throughput 16S rRNA gene sequencing and the sequence‐based function prediction with Tax4Fun. Of the 32 phyla detected (Proteobacteria class), 13 were dominant: Acidobacteria, Alphaproteobacteria, Actinobacteria, Betaproteobacteria, Bacteroidetes, Verrucomicrobia, Gammaproteobacteria, Deltaproteobacteria, Gemmatimonadetes, Firmicutes, Nitrospirae, Chloroflexi, and Planctomycetes (relative abundance >1%). We found no evidence that the diversity and composition of bacterial communities were significantly different among different cultivars, but the abundance of some dominant bacterial groups that have N‐fixation potentials (at broad or fine taxonomic level) and predicted abundances of some enzyme‐encoding nitrogenase genes showed significant across‐cultivar differences. The nitrogenase genes were notably abundant in Florigraze and Latitude soils while remarkably lower in Arbook and UF_TITO soils when compared with other cultivars, indicating different nitrogen fixation potentials among different cultivars. The findings also suggest that the abundance of certain bacterial taxa and the specific function bacteria perform in ecosystems can have an inherent association. Our study is helpful to understand how microbiological responses and feedback to different plant genotypes through the variation in structure and function of their communities in the rhizosphere.