Leptospirosis is a worldwide zoonosis, characterized by a complex ecological cycle, including the environment, wild and domestic animals, and humans. It is also considered a water-borne infection. Although a solid body of data is available for more conventional hosts, limited information is available on neglected species such as reptiles, especially Testudinidae, both captive and free-ranging. Molecular and serological tests were performed to detect Leptospira spp. in Testudo spp. in Italy. Leptospiral DNA was detected using cloacal swabs collected from 116 individuals (54 free-ranging and 62 captive tortoises) by polymerase chain reaction (PCR). Unfortunately, because of the presence of PCR inhibitors, swabs from only 50 animals were eligible for analysis. A serological microagglutination test was performed on 121 Testudo spp. (7 free-ranging and 114 rescued tortoises). Sixteen individuals were tested using both assays, and the remainder were tested with one or the other. Ten out of 50 (20%) amplifiable cloacal swabs were positive for Leptospira DNA, the identity of which was confirmed by sequencing two amplicons. Two out of four repeatedly sampled animals only tested positive in the autumn. Seroprevalence was relatively low (0.8%, 1/121), at low titer (1:100). No positivity was recorded among free-ranging animals. Although these results do not provide a complete epidemiological snapshot of leptospirosis in chelonians, they suggest that tortoises are susceptible to pathogenic Leptospira. Further data are needed to determine the role of the captive environment as a possible predisposing factor and to assess the role of these reptiles in the ecology of leptospirosis.