Tehran University of Medical Sciences Journal of Arthropod-Borne Diseases 2322-1984 18 3 2024 09 30 197 217 Alireza Barmaki Department of Vector Biology and Control of Diseases, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran, Department of Public Health, School of Public Health, Qazvin University of Medical Sciences, Qazvin, Iran Yavar Rassi Department of Vector Biology and Control of Diseases, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran, Arthropod-Borne Diseases Research Center, Ardabil University of Medical Sciences, Ardabil, Iran Azad Absavaran Department of Vector Biology and Control of Diseases, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran Amir Ahmad Akhavan Department of Vector Biology and Control of Diseases, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran Eslam Moradi-Asl 3Arthropod-Borne Diseases Research Center, Ardabil University of Medical Sciences, Ardabil, Iran, Department of Public Health, School of Public Health, Ardabil University of Medical Sciences, Ardabil, Iran Alireza Zahraei-Ramazani Department of Vector Biology and Control of Diseases, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran Sayena Rafizadeh Ministry of Health and Medical Education, Tehran, Iran 2023 12 12 2024 04 06 Background: Visceral leishmaniasis, commonly known as kala-azar, and prevalent in more than 70 countries and sev­eral regions of Iran. It is one of the main diseases transmitted by sand flies. In this work, geometric morphometrics and DNA barcoding were employed as novel techniques to enhance the diagnos­tic tools used in this study. Methods: Phlebotomus perfiliewi transcaucasicus, Phlebotomus major s.l., and Phlebotomus tobbi caught from three districts in the Ardabil Province, northwest of Iran. The right wings of 286 female sand flies were analyzed using geo­metric morphometric (GM) tools. Additionally, the COI gene was isolated from each of the three species, amplified using universal primers, and sequenced through the DNA barcoding method for classification. This sequencing data was then formatted to generate morphometric analyses. Results: The landmarks with the most variations were found in sets 10, 12, 13, and 14, whereas the first set's landmarks at 1 and 11, along with those from the second set at positions 2, 3, and 5 exhibited the greatest variations. Analysis of the size and shape variations in the wings indicates the presence of distinct populations (P< 0.05). Furthermore, the DNA barcoding results not only confirmed the findings from the geometric morphometric analysis but also revealed both interspecific and intraspecific distances. Conclusion: This study was the first attempt to assess whether wing geometry morphometrics, combined with DNA barcode techniques, can effectively distinguish the three mentioned species in the studied areas. Furthermore, the identi­fication of Phlebotomus neglectus in this area prompted recommendations for additional research. https://jad.tums.ac.ir/index.php/jad/article/view/1704 https://jad.tums.ac.ir/index.php/jad/article/download/1704/666