This study was determined the general combining ability (GCA) of parents and specific combining abilities of their hybrid combinations. Furthermore, hybrid vigor of F1 was evaluated by investigating genetic structure of the lines and testers. A total of 28 F1 hybrids (4 lines and 7 testers) were used as experimental material, which were developed from line × tester method. Investigated traits, except fiber length were influenced by non-additive gene effect in the created populations. Heterosis values for seed cotton weight per boll, earliness ratio, ginning percentage, fiber length and micronaire were positive, while seed cotton yield was negative. Gloria, TMN 199 and ZN 1018 proved the best parents for seed cotton yield, while TMN 199, GW 2357 and TMN 170 were best for earliness ratio. Similarly, GW 2357 and Gloria were best for seed cotton weight per boll, whereas Ipek 607 proved best for seed index. Likewise, TMN 199, TMD 139 ZN 1018 were best for ginning percentage, while Gloria, Flash, Ipek 607 and UA 48 proved best for fiber length. In the same way, Ipek 607 and TMD 139 were best parents for fiber fineness, whereas Gloria and UA 48 proved best for fiber strength. These parents had the highest overall combining abilities for respective traits. The hybrid combinations, i.e., TMN 199 × Ipek 607, TMN 199 × UA 48, Flash × GW 2357, Gloria × Ipek 607, Gloria × UA 48 and ST 468 × ADN 712 had the highest combining abilities for seed cotton yield. Similarly, TMN 199 × TMN 170 exhibited higher combining ability for earliness ratio, whereas Flash × GW 2357 and Gloria × Ipek 607 had better combining ability for seed cotton weight per boll. Likewise, TMN 199 × GW 2357 and Gloria × ADN 712 resulted in higher combining ability for seed index, while Gloria × TMD 139, TMN 199 × TMN 170, Flash × ZN 1018 and ST-468 × TMN 139 proved better for ginning percentage. Nonetheless, Flash × ADN 712 and ST-468 × Ipek 607 had better combining ability for fiber length, whereas ST-468 × Ipek 607 exhibited better combining ability for fiber micronaire. In the same way, TMN 199 × UA 48, Flash × Ipek 607, ST-468 × GW 2357 and ST-468 × UA 48 had better combine ability for fiber strength. The study identified the most promising hybrids for respective traits and overall combining abilities.