After two cycles of marker-assisted breeding on three loci, lines with transgressive segregation of 8.22-9.32 % protein content were developed based on four original soybean parents with 35.35-44.83 % protein content. Marker-assisted breeding has been an innovative approach in conventional breeding, which is to be further demonstrated, especially for quantitative traits. A study on continuous transgressive breeding for seed protein content (SPC) in soybean using marker-assisted procedures is reported here. The SPC of the recombinant inbred line (RIL) population XG varied in 38.04-47.54 % under five environments with P 1 of 35.35 %, P 2 of 44.34 % and total heritability of 89.11 %. A transgressive segregant XG30 with SPC 45.53 % was selected for further improvement. The linkage mapping of XG showed its genetic constitution composed of five additive QTL (32.16 % of phenotypic variation or PV) and two pairs of epistatic QTL (2.96 % PV) using 400 SSR markers with the remnant heritability 53.99 % attributed to the undetected collective of minor QTL. Another transgressive segregant WT133 with SPC 48.39 % was selected from the RIL population WT (44.83 % SPC for both parents). XG30 and WT133 were genotyped on the three major additive QTL (Prot-08-1, Prot-14-1 and Prot-19-2) as A 2 A 2 B 2 B 2 L 1 L 1 and A 1 A 1 B 1 B 1 L 2 L 2 , respectively. From WT133×XG30, surprising transgressive progenies were obtained, among which the recombinants with all three positive alleles A 2 _B 2 _L 2 _ performed the highest SPC, especially that of Prot-08-1. The five F 2-derived superior families showed their means higher than the high parent value in F 2:3 and F 2:4 and more transgressive effect in F 2:5:6, with the highest as high as 54.15 %, or 4.82 and 9.32 % more than WT133 and its original high parent, respectively. This study demonstrated the efficiency of marker-assisted procedure in breeding for transgressive segregation of quantitative trait.