Worldwide, soybean cyst nematode (SCN, Heterodera glycines Ichinohe) is the most destructive pathogen of soybean [Glycine max (L.) Merr.]. Crop losses are primarily mitigated by the use of resistant cultivars. Nematode populations are variable and have adapted to reproduce on resistant cultivars over time because resistance primarily traces to two soybean accessions, Plant Introduction (PI) 88788 and Peking. Soybean cultivar Hartwig, derived primarily from PI437654, was released for its comprehensive resistance to most SCN populations. A synthetic nematode population (LY1) was recently selected for its reproduction on Hartwig. The LY1 nematode population currently infects known sources of resistance except soybean PI567516C; however, the resistance to LY1 has not been characterized. The objective of this study was to identify quantitative trait loci (QTLs) underlying resistance to the LY1 SCN population in PI567516C, identify diagnostic DNA markers for the LY1 resistance, and confirm their utility for marker-assisted selection (MAS). Resistant soybean line PI567516C was crossed to susceptible cultivar Hartwig to generate 105 recombinant inbred lines ($F_2$-derived $F_5$ families). QTLs were mapped using simple sequence repeats (SSRs) covering 20 Linkage Groups (LGs) and three diagnostic markers, Satt592, Satt331, and Sat_274, were identified on LG O. These markers have a combined efficacy of 90% in identifying resistant lines in a second cross that has been generated by crossing a susceptible cultivar 5601 T with resistant PI567516C. $F_2$-derived $F_4$ segregating population was used in MAS to identify resistant lines.