Mutagenesis is used to study gene function and obtain new genetic resources for plant breeding. Soybean is an important oil crop in the world. Thus, to find new genetic resources, a mutation population was developed from the soybean cultivar Pungsannamul using 0.3% ethyl methane sulfonate. The variation of fatty acids was then evaluated among 892 M4 generation mutant lines selected from 3,774 mutant lines. While the wild type Pungsannamul showed 11.6, 3.4, 23.8, 53.3, and 7.8% for palmitic, stearic, oleic, linoleic and linolenic acid, respectively. the fatty acid variations in the mutant lines ranged from 7.4 to 19.7%, 2.2 to 13.0%, 14.7 to 49.0%, 31.8 to 63.9%, and 3.9 to 15.9% with an average of 10.8, 3.8, 25.3, 52.0, and 8.1% for palmitic, stearic, oleic, linoleic and linolenic acid, respectively. Thus, two mutation lines with higher plamitic acid, PE1542 (17.1%) and PE3058 (17.0%), one line with lower stearic acid, PE2166 (1.9%), one line with higher stearic acid, PE977 (12.7%), two lines with higher oleic acid, PE450 (44.4%) and PE2742 (47.7%), and two lines with lower linolenic acid, PE594 (4.6%) and PE1690 (3.7%), were selected from this study. The newly selected fatty acid variants will be good genetic sources for gene function analyses and breeding soybean varieties with altered fatty acids for various industrial and human food applications.