Soybean lipoxygenase (LOX) as an anti-nutritional factor can catalyze the enzymatic reaction of unsaturated fatty acids, resulting in an unpleasant beany taste negatively affecting the nutritional quality in soybean products. In this study, we cloned three lipoxygenase mutant genes lox1, lox2 and lox3, and analyzed their sequence variations among three genes in the near-isogenic lines (Suzuyutaka and Century) and cultivars of lipoxygenase mutant. The functional markers for LOX mutants (lox1-303 bp, lox3-266 bp, lox2-KASP, lox3-KASP) were developed and checked using agarose gel electrophoresis, capillary electrophoresis and KASP labeling techniques, followed by marker-assisted identification of their LOX genotypes in cultivars and breeding lines. Based on both of molecular marker and colorimetric methods, 25 soybean lines of LOX2 mutant (lox2) were identified. There was no significant difference in plant height, yield per plant, protein and oil content between LOX mutants and wild types within the population. Out of them, six soybean lines showing high yield and seed quality traits were identified. Collectively, this study provided the reliable functional markers and elite breeding lines in soybean molecular breeding for low beany flavor.