Plant expansins are a group of extracellular proteins thought to affect the quality of cotton fibers. Previous expression profile analysis revealed that six Expansin A genes are present in cotton, of which two(GhExp1 and GhExp2) produce transcripts that are specific to the developing cotton fiber. To identify the phenotypic function of Exp2, and to determine whether nucleotide variation among alleles of Exp2 affects fiber quality, candidate gene association mapping was conducted. Gene-specific primers were designed to amplify the Exp2 gene. By amplicon sequencing, the nucleotide diversity of Exp2 was investigated across92 accessions(including 7 Gossypium arboreum, 74 Gossypium hirsutum, and 11 Gossypium barbadense accessions) with different fiber qualities. Twenty-six SNPs and seven InDels including 14 from the coding region of Exp2 were detected, forming twelve distinct haplotypes in the cotton collection. Among the 14 SNPs in the coding region, five were missense mutations and nine were synonymous nucleotide changes. The average SNP/InDel per nucleotide ratio was 2.61%(one SNP per 39 bp), with 1.81 and 3.87% occurring in coding and non-coding regions, respectively. Nucleotide and haplotype diversity across the entire Exp2 region was 0.00603(π) and 0.844, respectively, and diversity in non-coding regions was higher than that in coding regions. For linkage disequilibrium(LD), the mean r2 value for all polymorphism loci pairs was 0.48, and LD did not decay over 748 bp. Based on132 simple sequence repeat(SSR) loci evenly covering 26 chromosomes, the population structure was estimated, and the accessions were divided into seven groups that agreed well with their genomic origin and evolutionary history. A general linear model was used to calculate the Exp2-wide diversity–trait associations of 5 fiber quality traits, considering population structure(Q). Four SNPs in Exp2 were associated with at least one of the fiber quality traits, but not with fiber elongation. The highest positive effect on UHML and STR was observed for haplotype Hap_6 of Exp2. There was a significant association of Exp2 with fiber quality traits. There were many haplotypes in the Exp2 region, of which the most favorable was Hap_6. The association between nucleotide diversity and these fiber traitssheds light on the gene’s potential contribution to the improvement of fiber quality, and should be useful to facilitate MAS programs in cotton. Plant expansins are a group of extracellular proteins thought to affect the quality of cotton fibers. Previous expression profile analysis revealed that six Expansin A genes are present in cotton, of which two (GhExp1 and GhExp2) produce transcripts that are specific to the developing cotton fiber To identify the phenotypic function of Exp2, and to determine whether the nucleotide variation among alleles of Exp2 affects fiber quality, candidate gene association mapping was conducted. Gene-specific primers were designed to amplify the Exp2 gene. By amplicon sequencing, the nucleotide diversity of Exp2 was investigated across92 accessions (including 7 Gossypium arboreum, 74 Gossypium hirsutum, and 11 Gossypium barbadense accessions) with different fiber qualities. Twenty-six SNPs and seven InDels including 14 from the coding region of Exp2 were detected, forming twelve distinct haplotypes in the cotton collection. Among the 14 SNPs in the coding region, five were missense mutations and nine w ere synonymous nucleotide changes. The average SNP / InDel per nucleotide ratio was 2.61% (one SNP per 39 bp), with 1.81 and 3.87% occurring in coding and non-coding regions, respectively. Nucleotide and haplotype diversity across the entire Exp2 region was 0.00603 (π) and 0.844, respectively, and diversity in non-coding regions were higher than that in coding regions. For linkage disequilibrium (LD), the mean r2 value for all polymorphism loci pairs was 0.48, and LD did not decay over 748 bp. Based on132 simple sequence repeat (SSR) loci evenly covering 26 chromosomes, the population structure was estimated, and the accessions were divided into seven groups that agreed well with their genomic origin and evolutionary history. A general linear model was used to calculate the Exp2-wide diversity-trait associations of 5 fiber quality traits, considering population structure (Q). Four SNPs in Exp2 were associated with at least one of the fiber quality traits, but not with fiber elongation. The higThere was a significant association of Exp2 with fiber quality traits. There were many haplotypes in the Exp2 region, of which the most favorable was Hap_6. The association between nucleotide diversity and these fiber traitssheds light on the gene’s potential contribution to the improvement of fiber quality, and should be useful to facilitate MAS programs in cotton.