ABSTRACTSelection of an appropriate hybridization method and use of molecular markers to study genetic purity of hybrids are important factors in crop improvement. In this study, our objective was to analyze two emasculation methods in finger millet and to present a strategy for the assessment of the genetic purity of F1 hybrids using molecular markers. Two parental lines of finger millet, PR 202 and IE 2606, were used for reciprocal crossing. Emasculation was performed with hot-water and hand methods; their efficiencies were compared. The hand method produced ~50-120 seeds per panicle and the hot-water method yielded ~30-90 seeds per panicle. The crossing efficiency was checked using molecular markers. The hot-water method was more efficient (52–80%) compared with the hand-emasculation method (16–40%). Simple sequence repeat (SSR), random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers were used to analyze the genetic variation in the parental lines. Twelve out of 28 RAPD markers, 4 out of 20 ISSR markers and 21 out of 101 SSR markers showed polymorphism between parental lines. The genetic purity was 16–80% based on RAPD and SSR markers. This study suggested that molecular markers can be utilized to compare the efficiency of emasculation methods and are highly useful for the identification of true hybrid seedlings in the early stages of growth.