Cotton is an important industrial crop worldwide and upland cotton (Gossypium hirsutum L.) is most widely cultivated in the world. Due to ever-increasing water deficit, drought stress brings a major threat to cotton production. Thus, it is important to reveal the genetic basis under drought stress and develop drought tolerant cotton cultivars. To address this issue, in present study, 319 upland cotton accessions were genotyped by 55,060 single nucleotide polymorphisms (SNPs) from high-density CottonSNP80K array and phenotyped nine drought tolerance related traits. The two datasets were used to identify quantitative trait nucleotides (QTNs) for the above nine traits using multi-locus random-SNP-effect mixed linear model method. As a result, a total of 20 QTNs distributed on 16 chromosomes were found to be significantly associated with six drought tolerance related traits. Of the 1,326 genes around the 20 QTNs, 205 were induced after drought stress treatment, and 46 were further mapped to Gene ontology (GO) term âresponse to stress.â Taken genome-wide association study (GWAS) analysis, RNA-seq data and qRT-PCR verification, four genes, RD2 encoding a response to desiccation 2 protein, HAT22 encoding a homeobox-leucine zipper protein, PIP2 encoding a plasma membrane intrinsic protein 2, and PP2C encoding a protein phosphatase 2C, were proposed to be potentially important for drought tolerance in cotton. These results will deepen our understanding of the genetic basis of drought stress tolerance in cotton and provide candidate markers to accelerate the development of drought-tolerant cotton cultivars.