Habitat fragmentation and destruction associated with the rapid urban and rural development of southeast Queensland presents an immediate threat to the survival of koala populations within this region. A sensitive method combining heteroduplex analysis (HDA) with temperature gradient gel electrophoresis (TGGE) was optimized to detect within-species variation in a mitochondrial DNA (mtDNA) control-region fragment, ≈ 670 bp in length, from the koala. Eight different haplotypes were characterized in koalas, of which four were novel. Analysis of mtDNA diversity in 96 koalas from five populations in southeast Queensland revealed that the number of haplotypes in a single population ranged from one to five, with an average within-population haplotype diversity of 0.379 ± 0.016, and nucleotide diversity of 0.22 ± 0.001%. Nucleotide divergence between populations averaged 0.09 ± 0.001% and ranged from 0.00 to 0.14%. Significant genetic heterogeneity was observed among most populations, suggesting that koala populations may be spatially structured along matrilines, although this may not be universal. The limited distribution of the central phylogenetic haplotype suggested the possibility of historical population bottlenecks north of the Gold Coast, while the presence of two highly divergent haplotypes at the Moreton site may indicate the occurrence of one or more undocumented translocation events into this area.