In the past decades, greenhouse effect elevated global average temperature, unexpected high temperature (HT) stress in summer limited the production of crop yield and agricultural development frequently. Cotton is the most important crop in providing a natural raw textile fiber for humans, which is mainly cultivated in summer and is reduction because of HT caused male sterility. However, the mechanism of HT caused male sterility in cotton is unclear. Thus, we need to screen HT-tolerant accessions and investigate mechanism of HT response in order to provide theoretical evidence and germplasm to solve this productive issue. We collected 517 natural upland cotton accessions and obtained the phenotypes under HT stress in multi-sites field experiments through self-developed biochemical index determination and deep learning methods. Totally, 39 accessions with high HT tolerance were identified, and we further study the HT responsive associated QTLs, eQTL regulatory maps and the casual genes through GWAS and TWAS. In addition, the DNA methylation, microRNA, H3K27me3, transcriptome, proteome basis in two accessions with distinct phenotype were analyzed, and the epigenetic regulation variation and the metabolic basis were elucidated and two kinds of kinase (CKI and HRK1) were identified that have potential roles in the anther. These works build a fast and efficient HT tolerant cotton identification technology system, and elucidate the mechanism of male reproduction organs responding to HT stress, and is conductive to create excellent polymeric germplasm for improving cotton HT tolerance breeding.