Aim: To report detection of leprosy in ocular tissue by histopathology and its confirmation by genetic analysis. Methods: Excised tissue from a clinically-suspected ocular leprosy patient was processed and analyzed histopathologically. The DNA from the paraffin-embedded tissue was extracted, an 85 A-C intergenic region of Mycobacterium leprae was amplified using specific primers and analyzed by conventional as well as real-time polymerase chain reaction (RT-PCR). Results: With periodic acid-Schiff-hematoxylin (PAS-H) staining the specimen showed presence of a thin fibrinous layer of inflammatory cells. The majority of the tissue was fibrovascular with extensive infiltration by histiocytes having reticulated cytoplasm. Modified PAS-H and acid-fast staining (AFS) showed the presence of several acid-fast organisms within the cytoplasm of histiocytes and mast cells. Conventional PCR showed a 250-bp DNA from excised conjunctival tissue, which was in agreement with the positive controls for M. leprae. Through RT-PCR, it was calculated that the suspected tissue had 44.68 pg of M. leprae DNA, which is 8937.06 genome copies of M. leprae. Conclusions: Presence of inflammatory cells and AFS bacilli in tissue presented a typical picture of leprosy. M. leprae DNA can be detected using RT-PCR in ocular tissues when acid-fast bacteria are seen in histopathological sections. And when the diagnosis of leprosy is inconclusive and acid-fast bacteria are seen, RT-PCR for M. leprae DNA could be used as a rapid confirmatory test to identify the presence of M. leprae and, therefore, the diagnosis of leprosy.