Soybean is an extensively utilized oilseed crop, and improved cultivars and cultivation efficiency of soybean have contributed to the increased use of soybean in edible oil applications. The food industry necessitates the development of soybean oil with an optimized balance of polyunsaturated and saturated fatty acids to meet both nutritional requirements and industrial applications. This study aimed to elucidate the protein structure and functional characterization of a novel allele of KASII-A derived from an EMS-induced mutant line and assess its potential as a genetic resource for developing soybean cultivars with elevated saturated fatty acid composition. Sequence variation in the KASII-A gene was evaluated for PE1544 (~ 16.1% palmitic acid composition), an EMS-induced mutant with high-palmitic acid. A single-nucleotide polymorphism was identified in the KASII-A gene of PE1544, resulting in an amino acid substitution from Gly309 to Asp309. Comparative analysis of three-dimensional protein structures revealed that Gly309 plays a critical role in stabilizing the catalytic residue in the KASII-A active site. Co-segregation analysis revealed that the novel allele was recessive to KASII-A and was associated with high-palmitic acid composition. Furthermore, we analyzed the F2 population derived from the cross between the high-stearic acid line with homozygous recessive sacpd-c allele and PE1544. The F2 progeny with both mutations exhibited a lower stearic acid composition compared to the single sacpd-c mutant. Notably, the F2 progeny with both mutations exhibited a similar ratio of polyunsaturated to saturated fatty acids (P/S index) compared to the single sacpd-c mutant. These findings suggest that KASII-A regulates the palmitic acid and stearic acid composition regardless of the total composition of saturated fatty acids in the single sacpd-c mutant. Comprehensively, the regulation of KASII-A in the single sacpd-c mutant is effective for the development of soybean oil with an ideal P/S index by regulating the content of palmitic and stearic acid while maintaining high-saturated fatty acids. These results suggest that the conversion of palmitic acid to stearic acid is impaired due to the loss-of-function of KASII-A, indicating that the novel allele of KASII-A plays a crucial role in this biochemical conversion in soybean.