Legumes Lessertia diffusa and Calobota sericea, indigenous to South Africa, are commonly used as fodder crops with potential for sustainable livestock pasture production. Rhizobia were isolated from their root nodules grown in their respective soils from the Succulent Karoo biome (SKB) in South Africa, identified and characterized using a polyphasic approach. Sequence analysis of the 16S rRNA gene confirmed all isolates as Mesorhizobium members, which were categorized into two distinct lineages using five housekeeping protein-coding genes. Lineage I included 14 strains from both legumes, while Lineage II comprised a single isolate from C. sericea. Differences in phenotypic traits were observed between the lineages and corroborated by average nucleotide identity analyses. While all strains nodulated their original hosts, strains from C. sericea failed to effectively nodulate L. diffusa and vice versa. Phylogenetic analyses of nitrogen fixation (nifH) and nodulation (nodA, nodC) loci grouped all strains in a single clade, suggesting that unique symbiotic loci determine nodulation of these legumes. We designated Lineage I and II as Mesorhizobium salmacidum sp. nov. (Ld1326