Chromosome segment substitution lines (CSSLs) are valuable genetic resources for quantitative trait loci (QTL) mapping of complex agronomic traits especially suitable for minor effect QTL. Here, 162 BC3F7-BC7F3 CSSLs derived from crossing two susceptible parent lines, soybean [Glycine max (L.) Merr.] 'Suinong14' (recurrent parent) x wild soybean (G. soja Siebold & Zucc.) ZYD00006, were used for QTL mapping of soybean cyst nematode (SCN, Heterodera glycine Ichinohe) resistance based on female index (FI) and cysts per gram root (CGR) through phenotypic screening and whole-genome resequencing of CSSLs. Phenotypic results displayed a wide range of distribution and transgressive lines in both HG Type 2.5.7 FI and CGR and demonstrated a higher correlation between CGR and root weight (R-2 = .5424) compared with than between FI and CGR (R-2 = .0018). Using the single-marker analysis nonparametric mapping test, 33 significant QTL were detected on 18 chromosomes contributing resistance to FI and CGR. Fourteen QTL contributing 5.6-15.5% phenotypic variance (PVE) to FI were revealed on 11 chromosomes, and 16 QTL accounting for 6.1-36.2% PVE in CGR were detected on 14 chromosomes with strong additive effect by multiple-QTL model (MQM) mapping. Twenty-five and 13 out of all 38 QTL identified for FI and CGR on 20 chromosomes were from ZYD00006 and Suinong14, respectively. The CSSLs with the combination of positive alleles for FI, CGR, and root weight exhibited low nematode reproduction. For the first time, QTL associated with CGR have been detected, and both FI and CGR should be considered for breeding purposes in the absence of strong resistance genes such as rhg1 and Rhg4.