Cotton is one among the few often cross-pollinated crops, where the achievement of commercial exploitation of heterosis is comparable to even that of  cross-pollinated crops like maize. The main bottleneck in exploitation of hybrid vigour in cotton is the complicated technique of seed production, which involves hand emasculation and pollination that makes hybrid cotton seed more costly. To improve the remunerative value of hybrid seed production and commercial cultivation of hybrid it is necessary to develop productive male sterile based hybrids. The study comprised 413 individual plants comprising of 164 A-line and B-line, 124 IPS of R-line, 13 hybrids, 3 cultivars, 4 wild species, 24 G. hirsutum germplasm lines, 80 RILs and 1 GMS line was carried out on identification and validation of molecular marker for fertility restoration during 2012-13 and 2013-14 at ARS, Dharwad farm. Twenty four molecular markers known to be associated with fertility restorer (10 RAPD. 7 SSR, 6 STS, and 1 TRAP) were studied. Out of these, 3 STS markers (Y1107, UBC 147 and UBC 607) were able to amplify only in 44 plants out of 124 belong to DR-7 (known as restorer). No amplification was observed in diverse genetic background of A-lines and their respective B-lines, wild species,  G. barbadense var. SBYF 425, G. arboreum var. DLSa 17, G. herbaceum var. Jayadhar, G. hirsutum germplasm lines ad Abadhita GMS line (sterile and its maintainer line). However, amplification by these three markers was recorded in 6 out of 80 RILs of DCH-32 hybrids.