Carotenoids are lipophilic isoprenoid pigments with essential roles in plants. While the cultivated allotetraploid cottons exhibit distinct mature anther coloration — yellow in Gossypium barbadense versus predominantly white in G. hirsutum — the genetic basis of this divergence remains unclear. The purpose of this study was to identify the genetic basis of anther-color variation in cotton (Gossypium) species. We firstly identified carotenoids as the primary pigments underlying yellow-anthers coloration. Comparative transcriptomics of anthers revealed that the carotenoid biosynthesis gene GbPSY4 was expressed as a key regulator in G. barbadense. Functional validation via tissue-specific expression, subcellular localization, in vivo enzymatic assays, and virus-induced gene silencing confirmed its role in carotenoid biosynthesis and yellow pigmentation. Genome-wide association studies in a G. hirsutum population revealed GhPSY4_At, an ortholog of GbPSY4, as the causal gene of anther-color variation. We conclude that PSY4-regulated carotenoid biosynthesis governs yellow pigmentation. Furthermore, a finding that G. hirsutum accessions with yellow anthers showed greater pollen viability under high-temperature stress than those with white anthers suggests that the same pathway that governs yellow pigmentation influences heat tolerance. PSY4 is a promising target for breeding stress-tolerant cotton varieties.