Soybean bacterial spot disease caused by Pseudomonas syringae pv. Glycinea which is a bacterial disease seriously affects soybean yield. Ten soybean germplasms and recombinant inbred lines (RILs) population were used to identify the resistant trait after inoculated with P. sg (P. sgneau001) in this study. High-density genetic mapping was obtained by specific length amplified fragment sequencing (SLAF-seq) of 149 RILs population which was derived from the crossing between Charleston and Dongnong594. The results indicated that 10 germplasm resources had four resistant germplasms included highly resistant cultivar Charleston, four susceptible varieties included Dongnong594 and two moderately resistant cultivars. Five quantitative trait locus (QTLs) were detected in RILs population by the composite interval mapping (CIM) method, and located on Linkage Group (LG) D1b (chromosome two), LG C2 (chromosome six) and LG H (chromosome 12), respectively. LOD scores ranged from 2.68 to 4.95 and the phenotypic variation percentage was from 6％ to 11％. Six candidate genes were detected, according to the result of gene annotation information. Four of them had relationship with protein kinase activity, protein phosphorylation and leucine rich repeat (LRR) transmembrane protein, which had high expression after inoculated with P. sg by qRT-PCR.