This study aimed to verify Leptospira spp. serovar infections in stray dogs in Itapema, Santa Catarina, Brazil. Serum samples were collected from 590 stray dogs and tested against 25 Leptospira spp. serovars using the microscopic agglutination test. Prevalence of anti-leptospiral antibodies against one or more serovars was 10.5%. The most frequent serovar was pyrogenes, positive in 26 (18.0%) samples, followed by canicola with 20 (13.8%) and icterohaemorragiae and copenhageni with 18 (12.5%, with antibody titers from 1:100 to 1:3,200). Significant prevalence (10.4 to 11.1%) was also detected against serovars castellonis, butembo, and grippothyphosa.