Proline, a critical osmoregulatory compound, is integral to various plant stress responses. The P5CS gene, which encodes the rate-limiting enzyme in proline biosynthesis, known as ∆1-pyrroline-5-carboxylate synthetase, is fundamental to these stress response pathways. While the functions of P5CS genes in plants have been extensively documented, their specific roles in cotton remain inadequately characterized. In this study, we identified 40 P5CS genes across four cotton species with diverse sequence lengths and molecular weights. Phylogenetic analysis of 100 P5CS genes from nine species revealed three subgroups, with Gossypium hirsutum closely related to Gossypium barbadense. Collinearity analysis highlighted significant differences in collinear gene pairs, indicating evolutionary divergence among P5CS genes in tetraploid and diploid cotton. Exon-intron structures and conserved motifs correlated with phylogenetic relationships, suggesting functional differentiation. Stress-responsive elements in P5CS promoters suggest involvement in abiotic stress. Expression analysis under salt stress revealed differential expression of GhP5CS genes, with GhP5CS1 emerging as a potential key regulator. Virus-induced gene silencing confirmed the pivotal role of GhP5CS1 in cotton’s salt stress response, as evidenced by increased salt sensitivity in the silenced plants. This study enhances our understanding of the functional diversity and stress response mechanisms of P5CS genes in cotton.