Eucalyptus morrisbyi is an endangered eucalypt, restricted to four populations on the island of Tasmania. The two main populations are separated by 20 km, occurring in the Risdon Hills and on Calverts Hill, and differ markedly in size and health. Although they are both in reserves, the small population at Risdon Hills has experienced a marked decline in the last two decades. The other two populations (Lumeah Point and Honeywood Drive) are very small and under threat because of urbanisation. They are close to the large Calverts Hill population and may be remnants of a once larger population in this area prior to clearing for agriculture in the 19th century. A hypervariable chloroplast marker and six nuclear microsatellites, used to quantify genetic diversity among and within populations, indicated marked genetic differences between the two main populations (Risdon and Calverts Hills), with virtually no sharing of chloroplast haplotypes and little sharing of microsatellite alleles among populations. Both of the main populations are clearly required to adequately conserve the genetic diversity in this species, whereas the Lumeah Point and Honeywood Drive populations are similar to the proximal Calverts Hill population. The two main populations showed equally high levels of genetic diversity (average H_E = 0.69) in the adult trees, using microsatellites, and little difference in inbreeding levels despite the large difference in population size. Analyses of 366 offspring from 9–11 trees from each main population revealed high outcrossing rates, little bi-parental inbreeding and high genetic diversity (average H_E = 0.65) in both seedling populations. This indicates that open-pollinated seed collections from these populations capture sufficient genetic diversity for ex situ conservation plantings. It is argued that the high genetic diversity maintained in the small Risdon Hills population is due to a combination of the longevity of its genotypes (possibly up to 1155–1523 years) through a well developed mechanism of vegetative regeneration from lignotubers, coupled with high outcrossing rates maintained by a strong self-incompatibility mechanism.