Fiber length and strength are the two most important traits in fiber quality of upland cotton (Gossypium hirsutum L.), and the understanding of their genetic basis is significant for breeding cultivars with high-quality cotton. We performed the genome-wide association study (GWAS) for fiber length and strength of association analysis group which comprises 315 upland cotton accessions grown in five different environments, through the mixed linear model (MLM). The results showed the presence of some differences in the phenotypic values of fiber length and strength, of high generalized heritability, with the fiber length variation coefficient ranging from 3.97% to 8.44%, and the fiber strength variation coefficient ranging from 7.85% to 11.26%. The analysis of variance for fiber length and strength showed highly significant effects of the genotype, environment, and genotype-environment interaction (P<0.001). Cluster analysis and population structure analysis showed that the 315 accessions could be divided into 2 groups. A total of 5 SNPs significantly associated with fiber length and/or strength were detected by GWAS, among which the locus D12_57032285 was significantly associated with both the fiber length and strength. The three loci significantly associated with fiber length were located on chromosomes A05, D11, and D12, respectively, which could explain 8.05%, 12.47% and 8.79% of the phenotypic variation, the elite allele types being A05_15144433 (AA), D11_24483544 (TT) and D12_57032285 (CC). The three loci significantly associated with fiber strength were located on chromosomes A08, D09 and D12, respectively, which could explain 9.03%, 7.94% and 7.90% of the phenotypic variation, the elite allele types being A08_84604654 (TT), D09_43463271 (TT) and D12_57032285 (CC). Through the analysis of gene expression patterns of two sets of different transcriptome data, 30 candidate genes that might be related to fiber development were selected. Through GO enrichment analysis and KEGG metabolic pathway analysis, it was found that the candidate genes mainly involved proteins or protein complexes and selectively and non-covalently interact with adenosine 5'-triphosphate (ATP), and the metabolic pathway was mainly the ribosomal metabolic pathway. The results can provide a theoretical basis for molecular genetic improvement of cotton fiber quality traits.