Australian freshwater fishes are a relatively species-poor assemblage, comprising a small number of Gondwanan lineages and a number of groups derived from repeated freshwater invasions by marine ancestors. In addition to being a comparatively small assemblage, they are both highly endemic and highly threatened. However, a comprehensive phylogeny for these taxa is lacking, which has hampered efforts to study their phylogenetic diversity, distribution of extinction risk, speciation rate, and rates of trait evolution. Here, we present a comprehensive dated phylogeny of 412 Australian freshwater fishes. We include all formally recognized freshwater species plus a number of genetically distinct subpopulations, species awaiting formal description, and predominantly brackish-water species. The phylogeny was inferred using maximum-likelihood analysis of a multilocus data set comprising six mitochondrial and three nuclear genes from 326 taxa. We inferred the evolutionary timescale using penalized likelihood, then used a statistical approach to add 86 taxa for which no molecular data were available. The time-tree inferred in our study will provide a useful resource for macroecological studies of Australian freshwater fishes, by enabling corrections for phylogenetic non-independence in evolutionary and ecological comparative analyses.