In this study, we performed a genetic diversity analysis using RAPD markers for some Vitex agnus-castus populations grown in Aydin, Turkey. Total genomic DNA isolation from the leaves of Vitex agnus-castus was performed using a commercial kit. Seven RAPD primers (OPA-02, OPA-05, OPA-13, OPA-15, OPA-16, OPA-18, OPA-20) were used to determine genetic diversity among populations. Polymerase Chain Reaction (PCR) was performed with all genomic DNA samples and primers. PCR products were run in agarose gel electrophoresis and visualized under UV light. The amplified products were scored as bands (1) and no bands (0) for all gel images and their matrix files were generated. A total of 36 characters were obtained from the primers. Phylogenetic relationships and genetic distances between the cultivars were calculated by using the PAUP* (Phylogenetic Analysis Using Parsimony and other methods) program. According to PAUP analysis, the closest genetic distances were between Çine pink flower and Çakmar purple flower, and Çakmar pink flower and Çakmar purple flower populations with a value of 0.05556; and the greatest genetic distance was between Çakmar pink flower and Köşk purple flower populations with a value of 0.36111. In the phylogenetic analysis obtained using UPGMA algorithms, the phylogenetic tree consisted of four groups. The results suggest that RAPD markers are useful tools for determining genetic relationships among Vitex agnus-castus genotypes.