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A quantitative trait locus (QTL) is a statically defined location of a gene governing that trait. QTL identification is the first step towards using marker-assisted selection (MAS) to introgress desirable QTL alleles into elite high-yield cultivars. Hundreds of high protein plant introductions (PIs) exist in the USDA germplasm collection and are a source of high protein alleles. Although 86 protein QTLs are currently listed in SoyBase, many are likely repeat discoveries of the same QTL(s), given the typical +/- 10 cM confidence intervals associated with QTL positions. Six germplasm accessions of maturity groups (MGs) II to IV that exhibited high seed protein (480 g kg-1 or more) were mated to a high-yielding cultivars of the same MG that exhibited normal seed protein (420 g kg-1 or less) to generate six F2 populations. A total of 240 individual F2 plants in each population produced F2:3 seed progenies that were phenotyped for seed protein content. Selective genotyping, or phenotypic tail analysis, was used to genotype only those F2:3 progenies occupying the lowest decile and the highest decile. A 1536-SNP locus assay chip was used for the genotyping. In the six mapping populations, eight protein QTLs with LOD scores greater than 3.0 were detected and mapped on five linkage groups using R/qtl. Significant QTLs on LG-C2 (Chromosome 6), LG-O (10), LG-B2 (14), LG-E (15), and LG-I (20) were detected. A review of the currently listed QTLs in Soybase (2010) indicated that no seed protein QTLs had been previously reported on LG-O (10). The new seed protein QTL discovered in this study in populations 1076, 1121, and 1122 is located on LG-O (10) near the two adjacent markers S19004 and S15265, and has an additive effect of 9.6, 7.9, and 6.5 g kg-1 greater seed protein, respectively. For improving the seed protein content in high yielding soybean cultivars, the accessions PI 437112A (1076), PI 398672 (1121), and PI 360843 (1122), which posses the high protein allele at this new LG-O (10) protein QTL, may be useful to soybean breeders.