Abstract Using PCR-based method, FbL2A gene was isolated from seven allotetraploid cotton (Gossypium hirsutum L. and G. barbadense L.) materials with different fiber quality and two diploid progenitors (G.herbaceum L. and G. richmondii U.). Based on single nucleotide polymorphisms (SNPs) of the FbL2A gene sequences in TM-1 and H-7124, the amplified products were digested by BstUⅠ through NEBcutter software online. And the gene was mapped on the chromosome 14 by Mapmaker v3.0b mapping software. In the accessions of upland cotton (G. hirsutum) with high fiber quality, the coding region SNPs with non-synonymous substitute were found, which may have phenotype effect.