We carried out DNA-hybridization comparisons among representatives of the major groups of Chiroptera to determine the phylogenetic position of the New Zealand short-tailed bat, Mystacina tuberculata. All analyses confirmed the noctilionoid affinity of this species suggested by an earlier serological study, with support from taxon jackknifing and at bootstrap levels of 98% or higher. However, a specific association with Noctilio was not found in more than 13% of the bootstrapped trees. The most precise of the thermal-stability indices employed (Tm, the median melting temperature of hybridized sequences) demonstrated a sister-group relationship of Mystacina to all noctilionoids, with Noctilio the first branch within Noctilionoidea but separated from the Mystacina lineage by a very short internode. Our determination of the timing of the divergence of Mystacina from noctilionoids is 54 myrbp. This estimate is based on independent indications that extant bat lineages began to diversify in the latest Cretaceous and is much earlier than the tentative estimate of 35 myrbp inferred from serology. Even if the diversification of all living bats occurred as early as 83 myrbp, as some authors have suggested, separation of Mystacinidae—n that basis, at 66 myrbp—could not have taken place soon enough for this taxon to be isolated on New Zealand before New Zealand separated from the rest of Gondwanaland. However, any of these dates would allow for the distribution of the noctilionoid-mystacinid common ancestor in South America, Australia, and Antarctica before the final sundering of Australia from Antarctica and for the divergence of Mystacinidae as a possible result of that event. This hypothesis is supported by the presence of fossil mystacinids in early and mid-Miocene deposits at Bullock Creek and Riversleigh, Queensland, showing that Mystacinidae had been resident in Australia from at least 25-20 myrbp. The most obvious scenario explaining the presence of Mystacinidae in New Zealand is therefore fortuitous dispersal from Australia across the Tasman Sea.