Six new microsatellite DNA loci are isolated from a genomic library of the sphenodontid reptile tuatara (Sphenodon) and presented here as a tool for identifying individuals for future paternity and kinship studies. These loci, in combination with four previously published loci, are sufficient to discriminate between clutch-mate siblings from Stephens Island and Brothers Island populations. These populations represent high and low levels of genetic diversity in tuatara populations respectively. An estimate of minimum number of fathers of each clutch found no evidence for multiple paternity in any clutch. These newly isolated loci complete the development of an array of genetic tools for use in tuatara to enhance ongoing conservation and management of wild, translocated and captive populations.