We estimated the timing of paleodrainage connections in the Colorado River Basin using mitochondrial deoxyribonucleic acid (DNA) sequence divergences among populations of the speckled dace, Rhinichthys osculus. Cytochrome b and ND4L sequences were analyzed by maximum likelihood methods to estimate phylogenetic branch lengths, which were calibrated to geological time with a fossil age estimate. We assume that heterogeneity in rate of evolution of mitochondrial DNA is caused in part by differences in body size, temperature, and correlated life-history traits; therefore, branch lengths are used directly to calculate rates of nucleotide substitution and ages of nodes on the phylogenetic tree. Rhinichthys osculus is estimated (by the corrected age of the oldest fossil) to have diverged from its sister species at 6.3 Ma. We estimate that speckled dace have been in the Colorado drainage for 3.6 m.y., and they have dispersed through the drainage and to former connectives, such as the Los Angeles Basin, in the past 1.9 m.y. Divergence among lineages of the upper and lower Colorado River drainages (above and below Grand Canyon) is estimated to have occurred ca. 1.9–1.3 Ma. Genetic divergence of allopatric lineages in the lower Colorado River drainage was accompanied by morphological adaptations to different stream gradients, but small genetic distances among these forms indicate recent gene flow and lack of reproductive isolation.