BackgroundCanine leishmaniosis caused by Leishmania infantum is a parasitic zoonotic disease transmitted by phlebotomine sandflies (Diptera: Psychodidae). Genus Phlebotomus is the biological vector in the Old World and Lutzomyia in the New World. The dog is the domestic reservoir host but other animals like the fox (Vulpes vulpes) and rodents are known to maintain the infection in both sylvatic and domestic cycles.MethodsTo identify the role of synanthropic rodents Mus musculus and Rattus norvegicus as reservoir hosts for Leishmania infantum natural infection, 30 rodents were captured under a trap rodent control program in two private dog shelters from Sintra and Sesimbra, located in the Lisbon Metropolitan Area, known to be endemic for canine leishmaniosis in Portugal. Tissue samples were screened for the presence of Leishmania amastigotes by qPCR and parasitological analysis.ResultsA total of 33.3% (9/27) of Mus musculus rodents revealed the presence of Leishmania spp. DNA while 29.6% (8/27) were positive in the parasitological analysis. Concerning Rattus norvegicus (n=3), one animal revealed infection only by parasitological analysis.ConclusionsOur results identified for the first time in Portugal the presence of Leishmania infection in both rodent species. As susceptible hosts, infected Mus musculus and Rattus norvegicus may increase the risk for dog and human infection in households and surrounding areas, enhancing the need for efficient rodent control measures in shelters and risk zones to prevent transmission of the infection.