The study conducted Line x Tester analysis using three female lines and eight male testers to assess heterosis, combining ability, and gene action in cotton. The experimental setup included eleven parents, resulting in twenty-four crosses and the standard check GN.Cot.Hy-14. Thirty-six genotypes were planted in a randomized block design with three replications at the Main Cotton Research Station, NAU, Surat during Kharif 2017. Analysis of variance revealed significant differences among parents and hybrids for various traits, indicating considerable genetic variability. Notably, certain crosses exhibited high per se performance and standard heterosis. Combining ability analysis highlighted the importance of both additive and non-additive gene effects in trait inheritance, with different traits showing varying degrees of genetic control. Furthermore, the study validated three GMS female lines, their fertile counterparts, and non-GMS original parental lines through SSR markers (BNL 1227 and NAU 2176), confirming the purity of genotypes and offering a quick and reliable means of identifying the genetic male sterility system governed by the ms5ms6 gene in cotton (G. hirsutum L.).