Cowpea (Vigna unguiculata L. Walp.) is indigenous to Africa, and highly valued for its N2-fixing trait and the nutritional attributes of its grain and leaves. The species' ability to establish effective symbiosis with diverse rhizobial populations gives it survival and growth advantage in N-limited environments. To explore the functional diversity and phylogenetic positions of rhizobia nodulating cowpea in Africa, nodules were collected from various cowpea varieties grown in soils from the Guinea savanna and Sudano-sahelian agroecologies of Northern Ghana, and from the lowveld and middleveld areas of Mpumalanga Province in South Africa. Box-PCR profiling and multilocus sequence analysis revealed the presence of diverse microsymbionts responsible for cowpea nodulation across the study sites. BOX-PCR amplifications yielded variable band sizes, ranging from 618 bp to 5354 bp, which placed the isolates in six major clusters (Cluster A-F). Phylogenetic analysis based on 16S rRNA, atpD, glnII, gyrB, rpoB, nifH and nodC genes revealed the presence of diverse Bradyrhizobium sp. closely related to Bradyrhizobium daqingense, Bradyrhizobium subterraneum, Bradyrhizobium yuanmingense, Bradyrhizobium embrapense, Bradyrhizobium pachyrhizi, Bradyrhizobium elkanii and novel Bradyrhizobium species in the soils studied, a finding that could be attributed to the unique edapho-climatic conditions of the contrasting environments. The test isolates exhibited distinct symbiotic efficiencies, and also induced variable (p ≤ 0.001) photosynthetic rates, leaf transpiration, total chlorophyll and shoot biomass accumulation on cowpea (their homologous host). Canonical correspondence analysis showed that the distribution of these microsymbionts was influenced by the concentrations of macro- and micronutrients in soils. The pairwise genetic distances derived from phylogenies and nodule functioning showed significant (p < 0.05) correlation, which suggests that local environmental factors played a major role in the cowpea-Bradyrhizobium symbiosis.