ABSTRACT: During 2019–2020, the Virgin Islands Department of Health investigated potential animal reservoirs of Leptospira spp., the bacteria that cause leptospirosis. In this cross-sectional study, we investigated Leptospira spp. exposure and carriage in the small Indian mongoose (Urva auropunctata, syn: Herpestes auropunctatus), an invasive animal species. This study was conducted across the three main islands of the U.S. Virgin Islands (USVI), which are St. Croix, St. Thomas, and St. John. We used the microscopic agglutination test (MAT), fluorescent antibody test (FAT), real-time polymerase chain reaction (lipl32 rt-PCR), and bacterial culture to evaluate serum and kidney specimens and compared the sensitivity, specificity, positive predictive value, and negative predictive value of these laboratory methods. Mongooses (n = 274) were live-trapped at 31 field sites in ten regions across USVI and humanely euthanized for Leptospira spp. testing. Bacterial isolates were sequenced and evaluated for species and phylogenetic analysis using the ppk gene. Anti-Leptospira spp. antibodies were detected in 34% (87/256) of mongooses. Reactions were observed with the following serogroups: Sejroe, Icterohaemorrhagiae, Pyrogenes, Mini, Cynopteri, Australis, Hebdomadis, Autumnalis, Mankarso, Pomona, and Ballum. Of the kidney specimens examined, 5.8% (16/270) were FAT-positive, 10% (27/274) were culture-positive, and 12.4% (34/274) were positive by rt-PCR. Of the Leptospira spp. isolated from mongooses, 25 were L. borgpetersenii, one was L. interrogans, and one was L. kirschneri. Positive predictive values of FAT and rt-PCR testing for predicting successful isolation of Leptospira by culture were 88% and 65%, respectively. The isolation and identification of Leptospira spp. in mongooses highlights the potential role of mongooses as a wildlife reservoir of leptospirosis; mongooses could be a source of Leptospira spp. infections for other wildlife, domestic animals, and humans. Author summary Leptospirosis is a zoonotic disease caused by bacteria of the genus Leptospira. To better understand local reservoirs and risk factors to humans and animals, during 2019–2020, the Virgin Islands Department of Health (VIDOH) investigated Leptospira spp. in association with the small Indian mongoose (Urva auropunctata, syn: Herpestes auropunctatus) across the three main islands of the United States Virgin Islands (USVI) (St. Croix, St. Thomas, and St. John). Mongoose blood and kidney samples were evaluated by laboratory testing: microscopic agglutination test, fluorescent antibody test (FAT), real-time polymerase chain reaction (lipl32 rt-PCR), and culture. Of 256 mongooses, 34% were exposed to Leptospira spp. To assess active infection in mongooses, kidney samples were examined resulting 5.8% (16/270) FAT-positive, 10% (27/274) culture-positive, and 12.4% (34/274) rt-PCR-positive. Validity was compared between test types showing higher positive predictive value for FAT and rt-PCR testing for predicting successful culture, the reference standard for diagnosis. Through the detection and isolation of Leptospira spp. in mongooses across the USVI, we identified mongooses as potential disease vectors to the local population and added to the limited data for the Caribbean region. Furthermore, a validity assessment of reference testing provides data to potentially inform future leptospirosis diagnostics.