The vital herb ashwagandha was farmed for commercial purposes in India. This study aimed to estimate the nature and amount of genetic variation in the 20 attributes in the 29 genotypes of ashwagandha in order to understand the correlations and character contributions to dry root weight/plant and alkaloid content with the selection of potential parents for ashwagandha crop improvement. Twenty-nine ashwagandha genotypes were assessed using a range of genetic metrics, including analysis of variance (ANOVA), genetic variability components, correlation, principal component analysis (PCA), and Mahalanobis D2. The ANOVA confirms a large amount of variability in the genotypes/varieties under study. Out of twenty the four traits, namely fresh leaf weight per plant (X8), dry leaf weight per plant (X9), seed yield per plant (X13), and dry root weight per plant (X16)—indicates the presence of additive gene action in the current study were highly heritable and had a high genetic advance mean value. Starch estimation, main root length, and root diameter were found to be positively and significantly correlated with dry root production per plant and total alkaloid content. The principle component analysis (PCA) results showed that the first four PCA accounted for the bulk (79.56%) of the differences. All genotypes are divided into four clusters based on the distances between and within clusters. Based on the findings mentioned above, it is recommended that genotypes from clusters IV (Poshita, CIMAP-Chetak), cluster II (AKAS-10, AKAS-11, NMITLI-101, and NMITLI-118), and cluster I (AKAS-02, IC-286632, and CIMAP Pratap) be chosen for a different hybridization program. Based on the multidisciplinary investigation, some of the genotypes with desired qualities have been found. They can be employed in upcoming breeding programs to increase alkaloid content and dry root yield.
