In this study, genetic diversity and phylogenetic analysis of some Vitex agnus-castus L. populations were conducted based on ISSR-PCR and chloroplast DNA trnL intron, and trnL-F sequence analyses. Vitex agnus-castus populations were detected in Aydın province, Turkey. Fresh leaf samples from the populations were collected and brought to the laboratory for genomic DNA isolation. 15 ISSR primers were used to determine the genetic diversity ofe Vitex agnus-castus populations. A total of 138 bands were obtained in ISSR analysis, 85 of which were polymorphic and 53 were monomorphic. Polymorphism rate was determined as 61.59 %. trnC, trnD, trnE, and trnF primers were used for PCR amplification of the chloroplast trnL intron and trnL-F region. A total of 138 bands were obtained by ISSR analysis. For trnL intron analyses, nucleotide lengths of 13 populations were between 508 and 516. The average nucleotide composition consisted of 38.5 % T, 18.3 % C, 27.5 % A and 15.7 % G. In trnL-F assays, the nucleotide lengths of the 13 populations ranged from 330 to 353. The average nucleotide composition consisted of 29.4 % T, 18.1 % C, 32.9 % A and 19.6 % G. The results of the phylogenetic trees constructed using some trnL intron and trnL-F sequences of Vitex doniana, Vitex trifolia, Vitex triflora, Vitex turczaninowii, Vitex queenslandica, Vitex axillariflora, Vitex rotundifolia and Vitex negundo species obtained from NCBI were compared. As a result of the study, polymorphisms were obtained at a rate of 61.59% from the ISSR analysis. In addition, the phylogenetic relationship between chloroplast trnL intron and trnL-F sequences of Vitex agnus-castus populations along with the other species was revealed.