The Indian cheetah was hunted to extinction by the mid-20th century. While analysis of 139 bp of mitochondrial DNA (mtDNA) has confirmed that the Indian cheetah was part of the Asiatic subspecies (Acinonyx jubatus venaticus), the detailed relationships between cheetah populations remains unclear due to limited genetic data. We clarify these relationships by studying larger fragments of cheetah mtDNA, both from an Indian cheetah museum specimen and two African cheetah, one modern and one historic, imported into India at different times. Our results suggest that the most recent common ancestor of cheetah mtDNA is approximately twice as ancient as currently recognised. The Indian and Southeast African (Acinonyx jubatus jubatus) cheetah mtDNA diverged approximately 72 kya, while the Southeast and Northeast African (Acinonyx jubatus soemmeringii) cheetah mtDNA diverged around 139 kya. Additionally, the historic African cheetah sampled from India proved to have an A. j. jubatus haplotype, suggesting a hitherto unrecognised South African route of cheetah importation into India in the 19th century. Together, our results provide a deeper understanding of the relationships between cheetah subspecies, and have important implications for the conservation of A. j. venaticus and potential reintroduction of cheetahs into India.