Soybean is one of the most important food crops worldwide.  Like other legumes, soybean can form symbiotic relationships with Rhizobium species.  Nitrogen fixation of soybean via its symbiosis with Rhizobium is pivotal for sustainable agriculture.  Type III effectors (T3Es) are essential regulators of the establishment of the symbiosis, and nodule number is a feature of nitrogen-affected nodulation.  However, genes encoding T3Es at quantitative trait loci (QTLs) related to nodulation have rarely been identified. Chromosome segment substitution lines (CSSLs) have a common genetic background but only a few loci with heterogeneous genetic information; thus, they are suitable materials for identifying candidate genes at a target locus.  In this study, a CSSL population was used to identify the QTLs related to nodule number in soybean.  Single nucleotide polymorphism (SNP) markers and candidate genes within the QTLs interval were detected, and it was determined which genes showed differential expression between isolines.  Four candidate genes (GmCDPK28, GmNAC1, GmbHLH, and GmERF5) linked to the SNPs were identified as being related to nodule traits and pivotal processes and pathways involved in symbiosis establishment.  A candidate gene (GmERF5) encoding a transcription factor that may interact directly with the T3E NopAA was identified.  The confirmed CSSLs with important segments and candidate genes identified in this study are valuable resources for further studies on the genetic network and T3Es involved in the signaling pathway that is essential for symbiosis establishment.