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Introduction and purpose: Human malaria is one of the widespread vector-borne diseases worldwide. Lots of efforts have been made to control and eliminate the vectors of the disease. Anopheles gambiae and Anopheles stephensi (A. stephensi) are the main vectors of malaria in Africa and Asia. The members of Anopheles gambiae complex contain the vector and non-vector species. A. stephensi has three biological forms with different vector capacities. The phenotypic and genotypic diversity has made it more difficult to identify vector populations and consequently the surveillance and control of malaria. The present study aimed to compare the morphological and molecular diagnostic characteristics of the two important malaria vectors in the two world continents. Methods: After searching, studying, and organizing published papers related to the subject, Anopheles gambiae and A. stephensi species were compared based on the morphological and molecular characteristics. Different morphological keys for the two species, the specimens from the insectarium of Urmia University of Medical Sciences, as well as the present species of national insectarium in Pasteur Institute of Iran, were used for morphological comparison. In addition, all the present sequences of five molecular markers, including COI, COII, D3, ITS2, and OBP1, were extracted from GenBank and analyzed using bioinformatics software. Results: Based on the obtained results of the present study, the number of ridges on Anopheles gambiae eggs was more than that on A. stephensi eggs. However, unlike A. stephensi in Anopheles gambiae, the number of ridges was not a diagnostic characteristic for the identification of Anopheles gambiae complex. There were four diagnostic characteristics in the larvae comparison of the two species and there were three different characteristics in adults. All the molecular markers were capable of separating the two species; however, ITS2 and D3, as well as COI, COII, and OBP1 markers were preferred for inter-species and intra-species comparisons, respectively. Conclusion: The combination of traditional diagnostic and new molecular methods can be simultaneously used in inter-species separation. As a result, in order to eliminate malaria in Iran and the countries covered by The World Health Organization Regional Office for the Eastern Mediterranean, it is recommended that the combination of morphological, molecular, and field epidemiological data can provide practical solutions for vector control programs. Obviously, none of these data exclusively respond to the needs of the mentioned programs. Furthermore, the coordination, design, implementation, and evaluation of applied projects, as well as executive actions, are necessary for the success of these programs regarding the elimination and eradication of malaria and other vector-borne diseases.