Cotton fiber quality is a persistent concern that determines planting benefits and the quality of finished textile products. However, the limitations of measurement instruments have hindered the accurate evaluation of some important fiber characteristics (such as fiber maturity, fineness, neps), which in turn has impeded the genetic improvement and industrial utilization of cotton fiber. Here, twelve single fiber quality traits were measured using Advanced Fiber Information System (AFIS) equipment among 383 accessions of upland cotton (Gossypium hirsutum L.). Also, eight conventional fiber quality traits were assessed by the High Volume Instrument (HVI) system. Genome-Wide Association Study (GWAS), linkage disequilibrium (LD) block genotyping and functional identification were conducted sequentially to uncover the associated elite loci and candidate genes of fiber quality traits. As a result, the pleiotropic locus FL_D11 regulating fiber length related traits was again identified in this study. More importantly, three novel pleiotropic loci (FM_A03, FF_A05, FN_A07) regulating fiber maturity, fineness and neps respectively were detected on the basis of AFIS traits. Numerous highly-promising candidate genes were screened out by integrating RNA-seq and qRT-PCR analyses, including the reported GhKRP6 for fiber length and newly identified GhMAP8 for maturity and GhDFR for fineness. The origin and evolution analysis of pleiotropic loci indicated that the selection pressure on FL_D11, FM_A03 and FF_A05 increased as the breeding period approached and the origins of FM_A03 and FF_A05 were traced back to cotton landraces. These findings reveal the genetic basis underlying fiber quality and provide insight into genetic improvements and textile utilization of fiber in G. hirsutum.