We present a study of the colonisation patterns of a tropical tree species among an island archipelago. Eucalyptus urophylla (S.T.Blake) is an economically important plantation species endemic to the volcanic slopes of seven islands in eastern Indonesia. In the present study, we investigated the geographical distribution of chloroplast DNA sequence variation in E. urophylla to gain insight into its historical seed-migration routes. DNA sequence data were obtained from 198 plants from which 20 haplotypes were identified. A moderate to high level of chloroplast genetic differentiation (G_ST = 0.581, N_ST = 0.724) and significant phylogeographic structure (N_ST > G_ST; P < 0.01) were observed, suggesting low levels of recurrent seed-mediated gene flow among the islands. The highest levels of haplotype diversity were observed on the eastern islands of Wetar and Timor. The two most westerly islands, Flores and Lomblen, were fixed for what appeared to be the ancestral haplotype. Chloroplast haplotype diversity therefore exhibited a decreasing trend from east to west in the species’ range, consistent with an east-to-west colonisation route across the seven islands. Environmental factors that may have contributed to the contemporary spatial distribution of chloroplast DNA haplotypes include island paleogeology, ocean currents, fluctuations in sea levels and possible hybridisation events.