To investigate the phylogenetic relationships and geographical structure among landlocked populations of the black mudfish, Neochanna diversus, mitochondrial DNA nucleotide sequence data were sampled from seven populations from the Waikato and Northland regions of New Zealand. The complete D-loop region was sequenced from 70 individuals, with 913 bp from the tRNA-pro end used in population and phylogenetic analysis. A tandem repeat array, which ranged in size up to 200 bp, was found in most populations at the 3' end of the D-loop that was not able to be aligned for analysis. Of the seven sites sampled, two from Northland exhibited significant sequence divergence from all other sites. There was also a clear distinction among remaining Northland sites and those from the Waikato. An additional 518 bp segment of the 16S region was sequenced from all sites and compared with the other New Zealand mudfish species, N. apoda, N. burrowsius and the Tasmanian mudfish Galaxias (Neochanna) cleaveri using Galaxias maculatus as an outgroup. Both D-loop and 16S sequence data provided strong evidence for a cryptic species of mudfish present in Northland. The significant genetic structure apparent in the black mudfish appears most probably to be attributed to geological conditions during the Pliocene, where peat wetlands became apparent in the Waikato while Northland consisted of disjunct 'islands'. Conservation and management of these populations must take into account the historical processes that have shaped these patterns of genetic diversity.