Abstract Background Leishmaniasis is a neglected parasitic disease caused by Leishmania spp., which is not endemic in Cuba. However, several factors (such as human activities, climate changes, and tourism) have led to an increase in the number of leishmaniasis cases in all regions, raising diagnosis and surveillance issues. We aim to present the retrospective analysis of 16 human cases suspicious of leishmaniasis, which were received during 2006–2016 for diagnosis at the Department of Parasitology from the Institute of Tropical Medicine Pedro Kourí, Cuba. Methods Clinical samples were collected and analyzed via different diagnostic assays, including direct smear, cultivation, histological analysis, and molecular analysis. Epidemiology and background of infection, clinical features, sex and age from each patient was recorded. Results From the 16 suspicious cases, 5 cases were confirmed for Leishmania infection, based on at least two positive results using different methods: PCR-based diagnosis [18S rRNA (5/5), hsp20 gene (4/5), hsp70 gene (3/5)], histopathology evaluation (2/3), parasite cultivation (2/3), or direct smears (2/3). L. braziliensis and L. mexicana were identified as the involving species in two cases, according to hsp70 PCR-RFLP protocols. Demographic and clinical features, as well as treatment and follow up, are described for every case. Conclusions The combination of parasitological and molecular methods allowed proper diagnosis of imported leishmaniasis cases in Cuba. The utility and advantages of molecular diagnosis assays in non-endemic countries like Cuba are discussed.