New Zealand's endemic frog species, Leiopelma hochstetteri, is a good model for studying the effects of population fragmentation on patterns of genetic diversity. L. hochstetteri inhabits streambeds that are becoming increasingly isolated due to urban and agricultural development. The potential for autosomal divergence between populations of L. hochstetteri is already apparent from the great extent of cytogenetic diversity within the species. Different populations exhibit variable numbers of B-chromosomes, ranging from none in some populations to as many as fifteen. Past studies on B-chromosomes in other taxa have indicated these karyological elements can affect gene flow between populations, resulting in genetic isolation and differentiation. This study investigated sequence variation in 600 bps of the mtDNA gene cytochrome b to determine the phylogenetic relationships between 17 populations of L. hochstetteri. The phylogeny and population structure was investigated using different methods: phylogenetic reconstruction, a minimum spanning network, and analysis of molecular variance. The sequence variation between L. hochstetteri and the outgroup, L. archeyi, was exceptionally high (20%) for a sister species. L. hochstetteri was found to be highly structured at the population level (64%, Φ = 0.740, p = 0.001) suggesting little or no gene flow between geographically close populations. Phylogenetic relationships above the population level were ambiguous. The presence or absence of B-chromosomes appears to have no, affect on population structure in L. hochstetteri. The molecular phylogeny indicacates B-chromosomes arose multiple times in L. hochstetteri, and that B-chromosomes evolved earlier than previously thought. Finally, in consideration of this new evidence on the phylogeny and karyology of L. hochstetteri, recommendations are made concerning the prioritisation of populations for conservation.