This study analyzed the phenotypic and genotypic characters of nodulating rhizobia isolated from two soybean cultivars, Kyushu 151 and Sachiyutaka, in the same field of the Yamaguchi Prefectural Technology Center of Agriculture and Forestry in Japan. The isolates were classified into groups using phenotypic characteristics, such as growth rate, color change on Bromothymol blue-containing yeast extract-mannitol agar (YMA) plates and colony morphology on YMA plates, and by genotypic characteristics, such as polymerase chain reaction–restriction fragment length polymorphism patterns of the 16S ribosomal RNA genes (16S rDNA) and the internal transcribed sequence (ITS) regions. In Kyushu 151, single phenotypic and genotypic groups were isolated from every nodule examined. In Sachiyutaka, plural strains belonging to distinct groups were obtained frequently from single nodules, indicating that multiple occupancy was established at high frequency. No fixed combination of the groups was found in the composition of multiple occupancy. An increase in the relative abundance of isolates belonging to Sinorhizobium fredii (Ensifer fredii) occurred concomitantly with the increase in the proportion of nodules with multiple occupancy. Nearly 60% of the isolates from Sachiyutaka belonged to S. fredii; 75% of them were obtained from nodules with multiple occupancy.