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Symbiotic nitrogen fixation (SNF) in legume crops plays a crucial role in world sustainable agriculture. To analyze the genetic basis of SNF in soybean, a natural population composed of 267 typical germplasms genotyped with 5403 SNP markers was inoculated with CCBAU45436, an elite rhizobium strain from China. Eleven traits related to SNF were evaluated under greenhouse and field conditions, and a genome-wide association study (GWAS) was conducted. In total, 20 SNP loci (consisting of 38 SNP markers) that were significantly associated with 11 SNF-related characteristics were identified on eight soybean chromosomes. Three SNP loci located on chromosome Gm17 were associated with shoot nitrogen concentrations in the two environments. Two other SNP loci located on Gm17 in the adjacent region were associated with high nodule numbers and nodule fresh and dry weights. Moreover, the elite alleles and genotypes were selected based on the phenotypes of different groups, which further demonstrated the reliability of these associated SNP markers. Other colocalized SNP loci linked with different SNF traits were also detected. More importantly, some candidate genes found at the associated SNP loci showed different expression levels between the different developmental stages of soybean nodules, two of which were further verified by qRT-PCR results. These SNP loci and candidate genes could be applied in marker-assisted selection in breeding or map-based gene cloning in soybean for future genetic improvement. [ABSTRACT FROM AUTHOR]