Leptospira spp. are zoonotic pathogens that may affect a variety of vertebrates. While they are typically associated with mammals, there are also reports of reptiles being exposed and infected with these spirochetes. To date, reports of this disease in wild chelonians suggest that exposure is not common; however, most of the reports evaluating this pathogen in reptiles are from outside of the United States. The purpose of this study was to estimate the seroprevalence of Leptospira spp. in Blanding's turtles (Emydoidea blandingii) found in an urban setting (DuPage County, Illinois). Serum samples from 29 adult (2 males, 27 females) and four subadult turtles were screened for the presence of Leptospira spp. antibodies against seven serovars common to Illinois using the microagglutination test (MAT). The seroprevalence in this study population was 93.5% (95% confidence intervals: 84.8–100). The high seroprevalence was unexpected based on previous reports in chelonians; however, pathogenic Leptospira interrogans had been reported in an area adjacent to the field site where the Blanding's turtles were captured. Because the MAT only characterizes exposure, it was not possible to determine if the turtles were infected or at risk of transmitting the pathogen. The findings did confirm that the pathogen was present in the habitat of these turtles. The results of this study suggest that Blanding's turtles may be useful as sentinels for characterizing the presence of Leptospira spp. in habitats throughout their range, but further research is needed to determine the role of these animals in the epidemiology of Leptospira spp.