Objective To use DNA detection methodologies to test for M. leprae in nine-banded armadillos inhabiting forested regions located around the cities and towns where leprosy patients have been identified. Design Ear lobe biopsies of 22 nine-banded armadillos were studied during a 2 year period. The biopsies were processed for DNA extraction and amplification by nested polymerase chain reaction (N-PCR) of a fragment of the high copy DNA locus of M. leprae known as R-LEP. Results Nine of the 22 (40.9%) armadillos evaluated showed positive signals for M. leprae. Sequencing confirmed that PCR products were identical to the corresponding region of M. leprae DNA. Conclusions In Colombia, South America, the consumption of and contact with the nine-banded armadillo (Dasypus novemcinctus) are common, ignoring the fact that this animal can host and be a possible zoonotic reservoir of Mycobacterium leprae, the causal agent of leprosy. This is the first study demonstrating that M. leprae is present in nine-banded armadillos in a region of Colombia using specific DNA detection. The possibility of leprosy transmission due to contact and consumption of armadillo meat or use of blood for therapeutic purposes should be further investigated.