Once common throughout the Indian subcontinent, the sloth bear (Melursus ursinus) is restricted to certain areas and is classified as vulnerable by the International Union for Conservation of Nature. Habitat loss and fragmentation is the primary reason for population decline, but there is a dearth of information about the effects of fragmentation on connectivity and genetic variation. Non-invasive DNA samples are commonly used to investigate connectivity and monitor populations in the wild. We developed a polymerase chain reaction (PCR) –based technique to identify sloth bears from non-invasively collected samples. Three sets of primers were designed to amplify short fragments of mitochondrial DNA to reduce false negatives, which is an issue with non-invasively collected DNA. The PCR success rate in both fresh and dry samples was high (88%). Failure to amplify DNA from other co-occurring mammals confirmed species specificity. This approach provides an efficient method to identify sloth bear samples using non-invasively collected DNA.