Conservation and restoration planning of extremely rare species relies on an understanding of the genetic diversity and population dynamics within a species to overcome potential inbreeding depression. Nānū or Nāʻū (Gardenia remyi H. Mann.) is an endemic tree native to the Hawaiian Islands and is one of more than 200 endangered plant species in Hawaiʻi with less than 50 individuals remaining in the wild. Efforts to understand the genetic diversity and connectivity between wild populations are foundational to conservation management plans, however little is known of the population structure of the species. In this study we utilize double digest restriction-site associated sequencing (ddRADSeq) on both historical herbarium specimens and samples from living ex situ collections to: (1) Test the hypothesis that we can capture genetic diversity in herbarium material of G. remyi using ddRADSeq, and (2) test the hypothesis that there are genetically distinct populations or subpopulation units among different Hawaiian islands. Usable sequencing data from thirty-seven samples of herbarium specimens collected between 1952 and 2017 and twenty wild sourced living collection samples were obtained representing all four islands where G. remyi is known to occur. Phylogenetic and population structure analysis revealed a monophyletic ingroup and a clear division between G. remyi samples of the northern island of Kauaʻi and those from the more southeastern younger islands of Molokaʻi, Maui and Hawaiʻi islands. The Kauaʻi samples were further split into a subpopulation from Southern Kauaʻi and the subpopulations from Northern Kauaʻi. Some admixed samples were detected. Our results are consistent with subpopulations of G. remyi, which needs to be considered in future conservation planning and breeding efforts to minimize inbreeding depression.