Abstract: Objective: The study aimed to compare the variance of constituents of volatile oil in the stems, leaves and flowers of Vitex negundo var. heterophylla (VNH). Methods: The volatile oil in the stems, leaves and flowers of VNH were extracted by hydrodistillation, respectively, and their constituents were analyzed and identified by GC-MS, and the differences in their chemical compositions were analyzed by principal component analysis (PCA) and cluster analysis. Results: Total of 83 volatile components were identified from different parts of the VNH, including olefins, alcohols, ketones, esters, etc. 14, 58 and 55 volatile components were detected from the stems, leaves, and flowers, accounting for 59.91%, 98.32% and 95.83% of the total volatile oils in each part, respectively. Only 8 volatile components were shared by three parts, whereas 4, 22 and 21 volatile components were unique to the stems, leaves and flowers, respectively. The differential components of the volatile oils from different parts of VNH were obtained by PCA, in which the signature volatile components in the stem were 2,4-bis (1,1-dimethylethyl)-phenol, 14-methyl-pentadecanoic acid, methyl ester, heneicosane, etc, and the trademark volatile components in the leaves were β-terpinene, elemol, caryophyllene oxide, etc, whereas the hallmark volatile components in the flowers were formed by sabenene, 2,3-dihydro-3-2-aminoethyl-5-methoxy-1,3- dimethyl-indole-2-one, citenamide, etc. As revealed by the results of the cluster analysis, the stems of VNH were clustered with the flowers and then with the leaves. Conclusion: The composition of the volatile oil in the stems, leaves and flowers of of VNH had certain variability, which would provide a scientific basis for the further medicinal or industrial exploitation of different parts of VNH.