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Not AvailableNot AvailaLipoxygenase (LOX) isozymes free soybean is critical for soy products with reduced off-flavour. In order to develope Lox3 free soybean, routine visual/spectrophotometric methods are performed on half seed for identification and egregation of null Lox3 plants from the population. They are cumbersome in distinguishing heterozygotes from homozygotes, involve the risk of damaging the embryo and delay the development of finished products by one generation. Single nucleotide polymorphism (SNP) and sequence tagged sites (STS) markers designed previously from the mutation in Lx3 gene of a triple-null Canadian soybean genotype OX948 could identify seedlings carrying null allele of Lx3 in a population derived from OX948. In the present investigation, the utility of these molecular markers in F2 population derived from a cross between Lox3-null donor genotype PI205085 and Indian soybean cultivar JS335 (Lox3+ve) has been validated. STS marker distinguished the homozygous dominant (Lx3Lx3), homozygous recessive (lx3lx3) and heterozygous (Lx3lx3) plants by generating PCR fragments of 476 bp, 396 bp and both the amplicons, respectively. SNP marker generated amplicon (296 bp) only in null allele carrying plants (Lx3lx3/lx3lx3) plants, and did not amplify homozygous dominant (Lx3Lx3). Using SNP marker, the target plants carrying null allele of Lx3 in F2 population were advanced to F6 generation. Finally, STS marker was employed to identify only homozygous recessive plants (lx3lx3). This resulted in the development of Lox3 free soybean genotypes, which can be used for development of double null (lx1lx1lx3lx3/lx2lx2lx3lx3) or triple null lipoxygenase (lx1lx1lx2lx2lx3lx3) soybean.bleNot Availabl