Abstract Genetic pathway of colonization of Lessepsian lizardfish Saurida undosquamis populations on the way from the Red Sea to Mediterranean Sea was examined. The lizardfish (Saurida undosquamis) samples were collected from the Red Sea (Jeddah) and Mediterranean Sea (Syrian Coast (Lattakia Port), Iskenderun Bay, Mersin Bay, Antalya Bay), and analyzed using mtDNA 16S gene region with mtDNA PCR-RFLP method with 6 restriction enzymes (BsurI AluI, Hin6I, RsaI, XhoI, EheI). A total of 16 haplotypes were detected from 150 individuals. AAAAAA haplotype was the most common occurred in all sampling sites present in 40% of individuals. The average haplotype diversity and genetic diversity within populations were 0.4579 and 0.009179, respectively. The average genetic diversity and genetic divergence between populations were calculated 0.025294 and 0.016115, respectively. In Monte Carlo (X2) pairwise comparisons highly significant differences (P < 0.001) between all populations were detected. S. undosquamis showed high genetic changes on the pathway of its colonization from south to northward, and there is lack of genetic migration between the Red Sea and Mediterranean