Genome-wide association studies (GWAS) of fiber quality traits of upland cotton were conducted to identify the single-nucleotide polymorphic (SNP) loci associated with cotton fiber quality, which lays the foundation for the mining of elite] cotton fiber gene resources and its application in molecular breeding. A total of 612 upland cotton accessions were genotyped using the ZJU Cotton Chip No. 1 40K chip array via the liquid-phase probe hybridization-based genotyping-by-target-sequencing (GBTS) technology. In the present study, five fiber quality traits, namely fiber length, fiber strength, micronaire, uniformity and elongation, showed different degrees of variation in different environments. The average coefficient of variation of fiber strength was the greatest, whereas the average coefficient of variation of uniformity was the least. Significant or extremely significant correlations existed among the five fiber quality traits, especially fiber length, strength, uniformity and elongation all being significantly negative correlated with micronaire. Population cluster analysis divided the 612 accessions into four groups: 73 assigned to group I, 226 to group II, 220 to group III and 93 to group IV. Genome-wide association studies of five fiber quality traits in five environments was performed and a total of 42 SNP loci associated with target traits was detected, distributed on 19 chromosomes, with eight loci associated with fiber length, five loci associated with fiber strength, four loci associated with micronaire, twelve loci associated with fiber uniformity and thirteen loci associated with fiber elongation. Of them, seven loci were detected in more than two environments. Nine SNP loci related to fiber length, fiber strength, uniformity and elongation were found on chromosome A07, seven loci related to fiber length, fiber strength, micronaire and elongation were detected on chromosome D01, and five loci associated with fiber length, uniformity and micronaire were detected on chromosome D11. The results from this study could provide more precise molecular markers and genetic resources for cotton breeding for better fiber quality in the future.