Simple SummaryLeishmaniasis is a group of parasitic diseases that affect humans and animals. Climate change and increased travel and migration have contributed to the spread of leishmaniasis in Europe, which may allow the introduction of new exoticLeishmaniaspecies or change the profile of known strains. Therefore, it is a priority to continue isolating and characterizingLeishmaniastrains from hosts. In this study, we analyzed and characterized twoLeishmaniaisolates (NAV and TDL) obtained from naturally infected mammals (dogs). We identifiedLeishmania infantumparasites, the main agents responsible for the disease in Spain and Europe. We focused on the analysis of growth rate, treatment response, infection capacity, and gene expression, comparing these isolates with the widely studied strainL. infantumBCN 150. Considering that these isolates showed different profiles, both NAV and TDL could be useful for in vitro and in vivo assays that might shed some light on the biology of the parasite.AbstractLeishmaniasis is spreading in Europe, especially in endemic countries such as Italy and Spain, in part due to ongoing climate change and the increase in travel and migration. AlthoughLeishmania infantumis the main agent responsible for this disease in humans and animals, other species and hybrids have been detected. This highlights the need to continue isolating and characterizingLeishmaniastrains from biological samples of infected hosts. In this study, we characterized the recently isolated parasitesL. infantumNAV andL. infantumTDL, obtained from naturally infected mammals (dogs), and we compared them with the widely distributed and studied strainL. infantumBCN 150. Both NAV and TDL promastigotes showed a slower growth rate than BCN 150 and were significantly more sensitive to amphotericin B and miltefosine. Furthermore, the expression of theCYCAgene (involved in cell cycle and proliferation) was significantly downregulated in NAV and TDL isolates. On the other hand,CYC6(implicated in treatment resistance) andAPG9(related to the recycling of protein under stress conditions and/or while undergoing a differentiation process and treatment resistance) levels were upregulated, compared to those measured in BCN 150. Both isolates displayed a higher infection capacity (>3 amastigotes per macrophage and >70% of infected macrophages) compared to controls (<2 amastigotes/cells and <50% of infected macrophages). Finally, a higher susceptibility to miltefosine treatment was observed in intracellular NAV and TDL amastigotes. In conclusion, TDL and NAV are novelLeishmaniaisolates that might be useful for in vitro and in vivo assays that will allow a better understanding of the parasite biology in Mediterranean areas.