In present study we evaluated the DNA damages and cytogenetic stability of transducted and non-transducted human dermal fibroblasts (HDFs) by enhanced green fluorescent protein (eGFP) lentiviral vector using karyotyping, comet assay, and molecular techniques. HDFs were isolated from human foreskin samples and eGFP-expressing lentiviral vector were transfected into HEK-293T cells to produce lentiviruses. Then, HDFs at passage 2 were transducted with concentrated eGFP lentivirus and transducted HDFs were detected by fluorescent microscope. The expression levels of cell cycle genes include two subunits of anaphase promoting complex (APC) in transducted and non-transducted HDFs were measured by quantitative real-time PCR and finally, karyotype test and comet assay was performed to evaluate the DNA damages and cytogenetic stability in both groups. The results of karyotype analysis were not showed any abnormalities in karyotype of transducted HDFs by eGFP in compared to normal cells. The mean values of alkaline comet assay parameters on non-transducted (normal cells), eGFP-transducted group and positive control (H₂O₂ treatment) were calculated by CaspLab software. The comparison of mean difference of comet assay parameters include tail length, comet length, tail moment, and Olive tail moment by T test between eGFP-transducted HDFs and other groups (positive control and non-transducted HDFs) were statistically significant (p£0.05). The alkaline comet assay on HDFs in eGFP-transducted group was showed small tail and indicated slight genetic damage compared with non-transducted group. Furthermore, the analysis of real-time PCR on expression of APC2 and APC7 genes in non-transducted HDFs compared with eGFP-transducted HDFs were not significant (p£0.05). These findings indicated that integration of lentiviral vectors in first passage of transducted HDFs could not disturb the DNA structure and create chromosome instability. So in genetic engineering and gene transformation these vectors in first passages are useful.