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Simple Summary: This study investigated the prevalence of Leptospira antibodies in stray dogs and cats in Milan, Italy. The results showed that 21.7% of the dogs tested seropositive for Leptospira antibodies, particularly the serovars L. icterohaemorrhagiae and L. Australis. In contrast, none of the cats tested seropositive. The study highlights the importance of ongoing serological surveillance in shelter environments to mitigate the zoonotic risk posed by leptospirosis. Leptospirosis is a widespread zoonosis recognised as a re-emerging infectious disease in both humans and dogs, yet the actual seroprevalence of Leptospira in pets in Italy is relatively unknown. The aim of this study was to evaluate Leptospira antibody prevalence in dogs and cats from a shelter by the microscopic agglutination test (MAT), the gold standard test in leptospiral serology, and to assess risk factors for Leptospira infection. This seroepidemiological study investigated the prevalence of leptospiral antibodies in a cohort of 106 dogs and 51 cats housed in a municipal shelter in Milan. Blood samples were collected from the animals during two sampling periods: spring/summer 2014 and autumn/winter 2016/2017. Eight serogroups were evaluated: L. Australis, L. Ballum, L. Canicola, L. Grippotyphosa, L. Icterohaemorrhagiae, L. Pomona, L. Sejroe, and L. Tarassovi. Antibody titres ranged from 1:100 to 1:6400. The results indicated that 21.7% of dogs had antibodies against serogroups L. Icterohaemorrhagiae and L. Australis, making them the most often found. Conversely, none of the cats showed any presence of antibodies. Seropositivity was higher in the spring/summer period (32.7%) than in autumn/winter (11.1%), and no statistically significant results were found regarding sex or age. These findings underscore the importance of ongoing serological surveillance and biosecurity measures in shelter environments to mitigate the zoonotic risk posed by leptospirosis. [ABSTRACT FROM AUTHOR]