Acceptance of the rough constancy of rates of molecular evolution, averaged over tens of millions of years, is widely used to date the splitting between taxa. However, for the study of speciation a hypothesis of rough constancy over tens of millions of years is of little use. In order to date the splitting of congeneric species within defined ranges of uncertainty, we need to know the variation of evolutionary rates over shorter periods of time. Such estimates of uncertainty are particularly useful if they apply to techniques of molecular comparisons that lend themselves to the assessment of intraspecific variation. We have measured protein divergence by electrophoresis and mitochondrial DNA differentiation by restriction fragment length polymorphism analysis in three pairs of sea urchin species believed to have resulted from the simultaneous fragmentation of ranges of marine species by the emergence of the Isthmus of Panama, about 3 million years ago. Transisthmian isozyme divergence in these pairs varies by an order of magnitude; mitochondrial DNA divergence, on the other hand, is equivalent in all pairs, suggesting that this molecule, assayed by endonucleases, can provide fairly accurate estimates of times since separation in the 3-million-year range.