Soybean is particularly known for accumulating saponins in its seeds. This study aimed to identify a causal gene to control an increase in Ab-γg saponin in PE1607 from an EMS-treated population of the soybean cultivar Pungsannamul. Segregation analysis in F₂ seeds verified that a single recessive allele controlled the increased Ab-γg saponin in PE1607. Bulk segregant analysis and mutant individuals identified the candidate region, containing the previously reported Sg-3 (Glyma.10G104700) gene, encoding a glucosyltransferase responsible for conjugating glucose as the third sugar at the C-3 position of the aglycone. NGS identified SNPs in the upstream of the Sg-3 gene, designated as the sg-3b allele. Expression analysis revealed that PE1607 exhibited a threefold decrease in Sg-3 expression in the hypocotyls compared to the Pungsannamul. Moreover, Sg-3 expressions significantly differed between the hypocotyls and cotyledons in developing seeds, with relatively low expression observed in the cotyledons. The results conclude that sg-3b allele may contribute to the reduced Sg-3 expression, resulting in an increase in Ab-γg saponin in PE1607. In addition, in the cotyledons, DDMP-βg and DDMP-βa saponins are present, containing rhamnose instead of glucose as the third sugar at the C-3 position of aglycone. This suggests that Sg-3, known as glucosyltransferase, does not significantly contribute to saponin biosynthesis in cotyledons.