We examined karyotypes of the endemic New Zealand reptile genus Sphenodon (tuatara) from five populations, finding a karyotype unchanged for at least one million years. Animals karyotyped were from five geographically distinct populations, representing three groups, namely S. guntheri, S. punctatus (Cook Strait group), and S. punctatus (northeastern North Island group). All five populations have a diploid chromosome number of 2n = 36, consisting of 14 pairs of macrochromosomes and four pairs of microchromosomes. Chromosomal differences were not found between the five populations nor between female and male animals, except for one animal with a structural heteromorphism. Similarity between Sphenodon and Testudine karyotypes suggests an ancestral karyotype with a macrochromosome complement of 14 pairs and the ability to accumulate variable numbers of microchromosome pairs. Our research supports molecular phylogenies of the Reptilia.