The soybean [Glycine max (L.) Merr.] pangenome has been studied and shown to be an invaluable resource for investigating structural variations (SVs), from which different genomic markers were successfully developed and employed for genome-wide association studies (GWAS). Among the SVs markers, gene presence-and-absence variations (PAVs) have been developed in soybean, but have not been widely utilized for association analyses. Here, we reported GWAS and haplotype analysis of seed protein and oil content for two diverse panels, comprised over 500 soybean accessions evaluated in multiple field environments using three marker datasets, whole genome sequence (WGS)-single-nucleotide polymorphisms (SNPs), 50 K-SNPs, and PAVs. The analyses identified new quantitative trait loci (QTL) for protein and oil content, along with the validation of previously reported QTL for these traits. This includes a well-studied QTL on chromosome (Chr.) 20 and another one on Chr. 05 for protein and/or oil. Importantly, this study is the first to report a new genomic locus for both protein and oil mapped to Chr. 08. Gene ontology annotations and expression profiles suggested candidate genes. Further analyses using haplotype-based markers led to the identification of multiple haplotype blocks encompassing candidate genes. Among these, Glyma.05G243400 on Chr. 05 and Glyma.08G109900 and Glyma.08G110000 on Chr. 08 were identified as promising targets. These genes can be incorporated into soybean breeding programs to enhance the selection of desirable protein and oil phenotypes through a haplotype-based breeding approach.