Australian freshwater fishes are a relatively species-poor assemblage, mostly comprising groups derived from older repeated freshwater invasions by marine ancestors, plus a small number of Gondwanan lineages. These taxa are both highly endemic and highly threatened, but a comprehensive phylogeny for Australian freshwater fishes is lacking. This has hampered efforts to study their phylogenetic diversity, distribution of extinction risk, speciation rates, and rates of trait evolution. Here, we present a comprehensive dated phylogeny of 412 Australian fishes. We include all formally recognized freshwater species plus a number of genetically distinct subpopulations, species awaiting formal description, and predominantly brackish-water species that sometimes enter fresh water. 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.