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Line x tester analysis using seven lines as females and five testers as males was carried out with a view to estimates heterosis, combining ability effects and nature of gene action along with identification and conformation of restorer line in CMS-R based hybrids in cotton. The experimental material comprised of twelve parents and their thirty five crosses along with standard check PKV Hy-4. The crosses GSHV-97/13 x AKH-7859 and GJHV-156 x AKH-03-8h appeared promising as they registered high per se performance with significant heterobeltiosis as well as standard heterosis with positive sca effects for seed cotton yield per plant, which indicated that these crosses could be exploited for commercial cultivation in respect of CMS-R base hybrid cotton. In present study, seven female lines, five male lines and 35 crosses screened through SSR marker with primer UBC 607500. Male lines and crosses had showed confirmatory results for fertility restoration. Thus, five lines AKH-03-8h, AKH-7859, DR-16, DR-8 and AKH-076R conformed as R-line and may be used as male lines in CMS-R based hybrid seed production programme.