Australian ants of the Myrmecia pilosula species complex include some individuals (in (M). croslandi) with the lowest possible metazoan chromosome number of (2 n=2). Others in this cluster of sibling species have much higher numbers, the known maximum being (2 n) = 32. Two species (M. pilosula and M. 'banksi') are believed on cytogenetic and morphological grounds to have hybridized over a long period. To investigate the phylogeny and age of this group relative to the congeneric outgroup species (M). gulosa, we sequenced part of the cytochrome (b) gene and the intergenic sequence between it and a primer anchored on the nearby tRNA SCN gene and analyzed the coding region using bootstrapped parsimony and neighbor-joining trees using the numbers of synonymous and nonsynonymous codons per site. The intergenic space demonstrated a profusion of repeated sequences, and only very closely related sequences (as judged by that for cytochrome b) showed detectable similarity at this almost (100 % \mathrm{~A}+\mathrm{T}) region. In agreement with predictions from karyotype studies, the phylogenetic analyses showed that (M). croslandi is the sister group to the other siblings; the time of separation of (M). croslandi from the rest of the pilosula group is unexpectedly ancient. Other relationships were poorly resolved, but the results suggest that (M). 'banski' and M. pilosula cluster together, as expected on cytogenetic grounds, and the tentative suggestion of close affinity of the M. pilosula samples and two "PB" samples supports derivation of (P B) from female (M). pilosula and male M. 'banksi.' (\odot 1995) Academic Press, Inc.