Meloidogyne javanica causing root-knot nematode in soybean is an important problem in soybean areas, leading to several yield losses. Some accessions have been identified carrying resistance loci to this nematode specie. In this study, a set of 317 soybean accessions were characterized for resistance to M. javanica. Genome-wide association study (GWAS) was performed using SNPs from genotyping-by-sequencing (GBS), and a region of 29.2 Kbp on chromosome 13 was identified. The haplotype analysis showed that SNPs were able to discriminate susceptible and resistant accessions, leading to 25 accessions sharing the resistance locus. Furthermore, 5 accessions may be new M. javanica resistance sources. The screening of the SNPs in the USDA soybean germplasm showed that several accessions previous reported as resistance to other nematodes also showed the resistance haplotype on chromosome 13. High levels of concordance among the phenotypes of Brazilian cultivars and the SNPs in chromosome 13 were observed. A in silico analysis of the mapped region on soybean genome revealed a presence of 5 genes with structural similarity with major resistance genes. The expression levels of the candidate genes in the interval demonstrated a potential pseudogene, and other two model genes up-regulated in the resistance source after pathogen infection. The SNPs associated to the region conferring resistance is a important tool for introgression of the resistance by marker-assisted selection in soybean breeding programs.