Vitex negundo L. is a potential medicinal and aromatic plant that is important for herbal medicine production. In this study, to evaluate the morphological and phytochemical variability among fifty accessions of V. negundo from South Gujarat of India. The result revealed that among the fifty accessions, VN 47 (Waghai, Dang) observed the highest plant height (5.50 m), plant girth (52 cm), number of primary branches (28) and crown spread (4.80 m²). And VN 20 (Mahuva, Surat) was identified as a promising accession for phytochemical parameters, exhibiting the highest Total Phenol Content (22.19 ± 0.04 mg GAE/100 g DW), Total Flavonoid Content (102.80 ± 1.47 mg QE/100 g DW), antioxidant activity (88.55 %) and essential oil content (0.193 %). Differences at the 5 % level (P < 0.05) were statistically significant. The principal component analysis of V. negundo accessions which revealed nine principal components that contributed to 76.6 % of the total variation in morphological and phytochemical parameters such as TFC, TPC, AA and EO have strong positive loading on PC1 and morphological plant height, canopy spread and number of primary branches were negatively correlated with PC1 and grouped in three main clusters, indicating that the observed diversity was largely driven by the influence of diverse genetic variability within accessions. This suggests superior accessions like VN 20 and VN 47 can be selected as chemotypes for further breeding programs, improvement and industrial application of V. negundo.