Ashwagandha is an important, medicinal, less exploited crop and most of its cultivated varieties are local cultivars. Information related to ashwagandha’s genetic diversity is very limited. Hybrid vigor exploitation may be useful in breaking its yield barrier at a commercial scale. A total of 45 F1 crosses were produced during Kharif 2017-18 by using a line x tester matting design, which included three testers along with 15 lines. A total of 65 genotypes were grown in an RBD design, with three replications in three different environments during Kharif 2018-19, and observations were recorded for 11 different yield-attributing traits that were responsible for the estimation of the magnitude of the heterosis. An analysis of variance showed that a valuable amount of diversity existed in the breeding material. Based on the study, three promising crosses viz., UWS-305 × UWS-10, UWS-305 × RVA-100, and UWS-301 × RVA-100 were identified as superior heterotic materials for the root yield at harvest, whereas for the total alkaloid content, three excellent hybrids viz., UWS-309 × RVA-100, UWS-305 × RVA-100, and UWS-314 × RVA-100 were reported. Out of the above crosses, the cross-combination of UWS-305 × RVA-100 showed a significantly positive and useful heterosis on a pooled basis. Hence, the above crosses may be gainfully utilized in breeding programs for ashwagandha.