Phylogenetic relationships among families within the order Atheriniformes have been difficult to resolve on the basis of morphological evidence. Molecular studies so far have been fragmentary and based on a small number taxa and loci. In this study, we provide a new phylogenetic hypothesis based on sequence data collected for eight molecular markers for a representative sample of 103 atheriniform species, covering 2/3 of the genera in this order. The phylogeny is calibrated with six carefully chosen fossil taxa to provide an explicit timeframe for the diversification of this group. Our results support the subdivision of Atheriniformes into two suborders (Atherinopsoidei and Atherinoidei), the nesting of Notocheirinae within Atherinopsidae, and the monophyly of tribe Menidiini, among others. We propose taxonomic changes for Atherinopsoidei, but a few weakly supported nodes in our phylogeny suggests that further study is necessary to support a revised taxonomy of Atherinoidei. The time-calibrated phylogeny was used to infer ancestral habitat reconstructions to explain the current distribution of marine and freshwater taxa. Based on these results, the current distribution of Atheriniformes is likely due to widespread marine dispersal along the margins of continents, infrequent trans-oceanic dispersal, and repeated invasion of freshwater habitats. This conclusion is supported by post-Gondwanan divergence times among families within the order, and a high probability of a marine ancestral habitat.