Abstract:Withania somnifera (L.) Dunal is one of the important medicinal plant native to India. In present study, four lines and seven testers were crossed in line × tester mating design to study the crossability success, extent of heterosis, nature of gene action, combining ability of parents and crosses. Experimental material comprising forty genotype including twenty-eight crosses, eleven parents and one standard control (GAA-1). Investigated traits in present study, during hybridization were number of emasculated flower bud, number of flower bud pollinated, number of berries formed, number of berries harvested, berry set percentage and during evaluation were days to flowering, days to maturity, plant height, number of berries per plant, seed yield per plant, root length, root diameter, fresh root yield per plant, dry root yield per plant, total alkaloid content. The study was carried out during rabi 2019-2020 and rabi 2020-2021 at Botanical Garden, Department of Genetics and Plant Breeding, C. P. College of Agriculture, Sardarkrushinagar Dantiwada Agricultural University, Sardarkrushinagr-385 506, Gujarat, India. Crossability success based on berry set percentage varied from 42.86% to 76.00% among the twenty-eight different crosses. Analysis of Variance for different traits under studied were presence of considerable amount of genetic variability among the parents and their crosses. The preponderance of non-additive gene action were reported for expression of all the characters based on GCA and SCA variance. The parents L4, T5 and T6 were possessed significant GCA effects for dry root yield and its component traits. Based on significant SCA effects, per se performance and heterosis; the crosses L1 × T6, L4 × T6, L3 × T4 and L4 × T7 were observed superior for dry root yield, its component characters and its quality.