Soybean seed oils contain approximately 23% oleic acid, and elevated amounts of oleic acid help prevent cardiovascular diseases and improve the quality of the oil. Chemically, it helps maintain the oxidative stability of oil; hence, soybean breeders primarily seek to increase its concentration for improved oil quality. As soybean seeds with mutant alleles of FAD2-1A and FAD2-1B genes have been reported to produce approximately 80% of oleic acid, a mutant population was developed from an ethyl methanesulfonate (EMS)-induced soybean cultivar (Pungsannamul). From this, a new mutant allele of FAD2-1A was identified using mutant lines with elevated oleic acid levels and the pooled-DNA sequencing method. This study identified PE529 as the allele with >40% oleic acid carrying the novel allele of the FAD2-1A gene. The single nucleotide polymorphism (SNP) in PE529 also induced the conversion from tryptophan to a premature stop codon at position 293 in the amino acid sequence (W293STOP). The inheritance analysis showed that the elevated oleic acids in PE529 were attributed to the fad2-1a W293STOP allele. In this study, seeds capable of producing approximately 80.0% oleic acid were identified from F2 populations where fad2-1a W293STOP and fad2-1b alleles were segregated. Hence, soybeans with novel alleles are useful genetic resources to improve soybean oil quality in breeding programs.