This study reports the genetic characterization of urban and rural populations of Phlebotomus (Phlebotomus) papatasi Scopoli (Diptera: Psychodidae) in Marrakech, Morocco. Using isoenzymatic analysis, four Moroccan populations were compared with other Mediterranean basin populations from Spain, Cyprus, and Syria. Morphological anomalies were noted in the male genitalia of 5.3% of the specimens collected from Marrakech area. Qualitative analysis of zymogram profiles revealed nine polymorphic enzymes (HK, PGM, PGI, 6PGD, MDH1, MDH2, ICD2, FUM, and ACO) and three monomorphic enzymes (ME, ICD1, and alphaGPDH). Genetic distances clearly separated the populations of western Mediterranean countries (Morocco and Spain) from eastern countries (Syria and Cyprus), but they could not be used to differentiate between urban and rural populations in Marrakech area.