Leptospirosis is a worldwide zoonosis caused by the pathogenic Leptospira spp. Canine and human leptospirosis sometimes occur on Amami Oshima Island, located in the Nansei Archipelago, southwestern Japan; however, information on the causative Leptospira spp. on this island is quite limited. This study aimed to investigate the molecular and serological characteristics of Leptospira spp. isolated from wild animals and a dog in Amami Oshima Island. We obtained seven Leptospira strains by culturing kidney tissues of wild animals, such as black rats (2), wild boars (3), and rabbit (1) as well as blood from a symptomatic dog. Using flaB sequencing and microscopic agglutination test with antisera for 18 serovars, the isolates were identified as Leptospira borgpetersenii serogroups Javanica (black rat), L. interrogans serogroup Australis (black rat and dog), and L. interrogans serogroup Hebdomadis (wild boar and rabbit). The sequence type (ST) of L. borgpetersenii serogroup Javanica was determined to be ST143 via multilocus sequence typing (MLST) using seven housekeeping genes. For L. interrogans, MLST and multiple-locus variable-tandem repeat analysis (MLVA) revealed identical ST and MLVA types in rat and canine isolates, whereas two STs and MLVA types were identified in wild boar isolates. The STs and MLVA types of rabbit and one of the wild boars were identical. Bacterial culture and flaB-nested polymerase chain reaction demonstrated a high rate of Leptospira infection in wild boars (58.3%, 7/12), whereas Leptospira spp. were detected in 4.8% of black rats (2/42). This study revealed diverse Leptospira genotype and serotype maintenance in wild mammals on Amami Oshima Island. MLST and MLVA indicated that black rats were a source of canine infection. Wild boars carry L. interrogans and are considered an important maintenance host because antibodies against serogroup Hebdomadis were detected in human and canine leptospirosis patients on this island.