Cultivated soybean (Glycine max [L.] Merr.) is a major global source of vegetable protein and edible oil, yet its production is severely constrained by soybean cyst nematode (SCN, Heterodera glycines), with race 3 being the most prevalent and destructive pathotype in China. Zhongpin 03-5373 (ZP03) is an elite Chinese soybean line characterized by stable resistance to SCN3 (race 3, HG type 0) and superior agronomic performance. Here, we report a near-gapless de novo genome assembly of ZP03 generated using PacBio Revio (HiFi) long-read sequencing combined with Hi-C chromatin interaction mapping. The assembled genome spans 1,067.68 Mb with a Contig N50 of 35.36 Mb, anchoring 95.24% of sequences onto 20 chromosomes. Comparative genomic analyses revealed an extensive structural variation (SV) landscape associated with SCN3 resistance in ZP03. A 393-bp deletion-type SV located in the promoter region of GmSNAP11 was identified and was associated with increased gene expression following SCN3 infection. This allelic variation may contribute to differential transcriptional regulation under pathogen challenge. Using an SV-based genetic map constructed from a ZP03×Zhonghuang 13 (ZH13) recombinant inbred line population, we further identified a novel QTL qSCN3-16 contributing to SCN3 resistance, which explaining 7.51% of the phenotypic variance (LOD=3.42). Within this locus, Glyma.16G045900 (SYP16), encoding a t-SNARE protein, was prioritized as the candidate gene. Haplotype analysis across 2,214 diverse soybean accessions demonstrated that a favorable promoter SV in SYP16 is significantly associated with enhanced SCN3 resistance. Together, these results highlight the critical role of structural variation in shaping soybean immune responses and provide high-resolution genomic resources and functional markers, specifically the novel QTL qSCN3-16, to support precision breeding of SCN3-resistant soybean cultivars.