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Soybean nutritional quality traits are generally quantitative traits and are regulated by multiple genes. At present, a large number of molecular markers related to quality traits have been located, but there were few available markers verified in soybean breeding populations. In this study, we selected 288 soybean varieties from Huang-Huai-Hai region and 19 wild/semi-wild soybean accessions as a natural population. We analyzed their protein, oil, fatty acid, and isoflavone contents by NIR, GC, and HPLC methods. We selected 18 SSR markers closely linked to quality traits to identify their genotypes by capillary electrophoresis method, and verify the molecular markers associated with nutritional quality traits using association analysis based on the Tassel 2.1 software. The results showed that there were three markers associated with oil content, three markers associated with the content of protein plus oil, one marker associated with palmitic acid, one marker associated with stearic acid, two markers associated with oleic acid, and two markers associated with linolenic acid, and the elite alleles of these loci were also investigated. Our study provided reliable molecular markers for soybean quality molecular breeding. [ABSTRACT FROM AUTHOR]