Beany flavor induced by three lipoxygenases (LOXs, including LOX1, LOX2, and LOX3)restricts human consumption of soybean. It is desirable to generate lipoxygenase-free new mutant lines to improve the eating quality of soybean oil and protein products. In this study, a pooled clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPRassociated protein 9 (Cas9) strategy targeting three GmLox genes (GmLox1, GmLox2, and GmLox3) was applied and 60 T0 positive transgenic plants were generated, carrying combinations of sgRNAs and mutations. Among them, GmLox-28 and GmLox-60 were gmlox1gmlox2gmlox3 triple mutants and GmLox-40 was a gmlox1gmlox2 double mutant. Sequencing of T1 mutant plants derived from GmLox-28, GmLox-60, and GmLox-40 showed that mutation in the GmLox gene was inherited by the next generation. Colorimetric assay revealed that plants carrying different combinations of mutations lost the corresponding lipoxygenase activities. Transgene-free mutants were obtained by screening the T2 generation of lipoxygenase-free mutant lines (GmLox-28 and GmLox-60). These transgeneand lipoxygenase-free mutants could be used for soybean beany flavor reduction without restriction by regulatory frameworks governing transgenic organisms.