Simple SummaryThe isolation of Leptospira is the most important test to assess infection in animal species. Several animals play a role as maintenance-host for specific serovars and in the last 30 years the incidence of leptospirosis has constantly increased in well-known reservoirs as well as in “unconventional” hosts. The emergence and the identification of Leptospira infection in such “unconventional” hosts could be related to several factors including problematic or inaccurate sampling modes during the Leptospira isolation, newly identified Leptospira strains, underestimated leptospirosis cases and climatic changes causing modifications of ecological niches. The aim of this review was to report the Leptospira isolations of the last 60 years from animals that could be considered “unconventional” hosts. Thus, the identification of “unconventional” hosts is crucial because they almost surely play an important role in the epidemiology of Leptospira infection.Leptospirosis is a re-emerging worldwide zoonotic disease. Even though the primary serological test for diagnosis and surveying is the microscopic agglutination test (MAT), isolation remains the gold-standard test to detect Leptospira infections. The leptospirosis transmission is linked to maintenance and accidental hosts. In the epidemiology of Leptospira some serovar are strictly related to specific maintenance hosts; however, in recent years, the bacterium was isolated from an even wider spectrum of species. The aim of this review is to report the isolation of Leptospira strains in animals which could be recognized as “unconventional” hosts, analyzing studies from 1960 to 2020 that highlighted the Leptospira isolation. This scientific literature aimed to provide evidence of infection in several animal species including of the Carnivora, Didelphimorphia, Rodentia, Cetacea, Cingulata, Afrosoricida, Chiroptera and Primate orders, as well as in Reptilia and Amphibia classes. In conclusion, the spreading of Leptospira is attention-worthy because the infection could occur in all the animal species ranging in a specific area. Further screening and isolations are needed to collect all necessary data to gain a complete understanding of leptospirosis epidemiology and its modifications.