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Background: Leishmaniasis is a neglected disease caused by different species of the protozoa Leishmania spp. Cutaneous lesions are the most common clinical manifestation. This disease is prevalent in tropical and subtropical areas, including the Mediterranean basin. In Spain, Leishmania (L.) infantum is the only endemic species, but imported cases are often diagnosed. Different classical parasitological methods can be performed for cutaneous leishmaniasis (CL) diagnosis; but currently molecular techniques serve as a relevant tool for the detection and characterization of Leishmania parasites. We aimed to evaluate clinical and epidemiological characteristics of CL diagnosed patients by real-time PCR in a tertiary hospital over a six-year period. Methodology/Principal findings: Clinical, epidemiological and microbiological data were retrospectively collected and analyzed. In our study, CL was confirmed in 59 (31.4%) out of 188 patients by real-time PCR, showing an increase over recent years: 11 cases of CL between 2014 and 2016 and 48 between 2017 and 2019. Real-time PCR was performed on skin swabs and/or biopsies samples, with a positivity of 38.5% and 26.5%, respectively. Results were 100% concordant when biopsy and skin swab were performed simultaneously. L. (L.) infantum was the most frequent species detected (50%), followed by L. (L.) major (45%) and Viannia subgenus (5%), which were detected only in imported cases. L. (L.) major was almost entirely detected in travelers/migrants from Morocco. Multiple and atypical skin lesions were more common in imported cases than in autochthonous cases (44.4% vs. 21.8%). Conclusions/Significance: An increase in both autochthonous and imported CL cases has been observed in past years in our hospital. Molecular techniques assist in improving CL diagnosis and characterization of the Leishmania species, mainly in imported cases. Author summary: Leishmaniasis is a neglected disease caused by different species of the genus Leishmania. This disease is prevalent in tropical and subtropical areas, including the Mediterranean basin. In Spain, the only endemic species is L. (L) infantum, although there are cases of imported leishmaniasis due to travel and migration of people from other endemic areas. Cutaneous lesions are the most common clinical form, but L. (L) infantum can also cause mucosal or visceral involvement. Although microscopic diagnosis and skin biopsy specimens are still used in routine diagnosis for cutaneous leishmaniasis, they are time-consuming and invasive, respectively. Currently, molecular techniques, such as real-time PCR, are very sensitive tools for the detection and identification of Leishmania parasites. In addition, the skin swab sample seems to be a non-invasive alternative for the diagnosis of cutaneous leishmaniasis. In the present work, we describe the clinical characteristics of 59 patients diagnosed by real-time PCR of cutaneous leishmaniasis, as well as the molecular epidemiology of the causative species. We show that there has been an increase in cutaneous leishmaniasis cases, both autochthonous and imported cases, over the study period. The presence of Leishmania species other than L. (L) infantum is also highlighted. [ABSTRACT FROM AUTHOR]