Soybean seed composition affects the utility of soybean, and improving seed composition is an essential breeding goal. Fast neutron radiation introduces genomic mutations resulting in novel variation for traits of interest. Two elite soybean lines were irradiated with fast neutrons and screened for altered seed composition. Twenty-three lines with altered protein, oil, or sucrose content were selected based on near-infrared spectroscopy data from five environments and yield tested at five locations. Mutants with significantly increased protein averaged 19.1–36.8 g kg−1 more protein than the parents across 10 environments. Comparative genomic hybridization (CGH) identified putative mutations in a mutant, G15FN-12, that has 36.8 g kg−1 higher protein than the parent genotype, and whole genome sequencing (WGS) of the mutant has confirmed these mutations. An F2:3 population was developed from G15FN-12 to determine association between genomic changes and increased protein content. Bulked segregant analysis of the population using the SoySNP50K BeadChip identified a CGH- and WGS-confirmed deletion on chromosome 12 to be responsible for elevated protein content. The population was genotyped using a KASP marker designed at the mutation region, and significant association (P < 0.0001) between the deletion on chromosome 12 and elevated protein content was observed and confirmed in the F3:4 generation. The F2 segregants homozygous for the deletion averaged 27 g kg−1 higher seed protein and 8 g kg−1 lower oil than homozygous wild-type segregants. Mutants with altered seed composition are a new resource for gene function studies and provide elite materials for genetic improvement of seed composition.