The pattern of variability in chloroplast DNA (cpDNA) of Eucalyptus globulus Labill. (Myrtaceae) was studied using 270 samples from southern Australia. Forty variable sequence characters were found, defining 105 haplotypes. Haplotypes were assigned to three major cpDNA clades based on their phylogeny. The pattern of cpDNA variation did not conform to subspecies boundaries; however, there was a strong geographic structure to the distribution of clades and haplotypes. One clade (JC) was geographically central and widespread and was found in 163 samples from Tasmania and continental Australia. Less-common clades occurred in more localised regions - southern (Js, 77 samples) and eastern (Jet, 12 samples). The distribution of the Jet and Js clades coincides with hypothesised glacial refugia in Tasmania, suggesting limited seed-mediated dispersal since deglaciation.