Luffa (Luffa cylindrica) is a plant from the Cucurbitaceae family that grows mostly in tropical and subtropical regions, as well as in most regions of Iran. In this research, the genetic diversity of nine native and non-native genotypes of L. cylindrica was investigated through the evaluation of the chloroplast trnH-psbA intergenic region (IGS). After sampling the young leaves, DNA extraction was performed by using the Dellaporta method, and PCR was conducted by using IGS intergenic region primers. After sequencing of the amplified products, their quality was determined using Chromas software and then aligned using ClustalW method by BioEdit and MEGA7 softwares. Next, the dendrogram of phylogenic relationships was drawn and the matrix of the difference and similarity of the sequences were determined. In the present research, by analyzing the relationships between studied samples, based on the trnH-psbA (IGS) marker, a strong intraspecies variation was observed in native and non-native L. cylindrical genotypes. The genetic distance matrix between the samples examined in this research ranged from 0 to 6.865 with an overall average distance of 2.53. The average value of synonymous and non- synonymous substitutions (dN/dS) for the IGS sequence was ds/dn = 0.68, which indicates positive and pure line selections in the process of natural selection of studied genotypes. The results of this research showed that trnH-psbA is a suitable marker for evaluation of the intraspecific diversity of luffa species.