The question of how new species arise has bedeviled biologists for a century and a half. His famous tome may have been titled “On the Origin of Species,” but Darwin had little idea of how one species was transformed into another. Recent years, however, have seen great attention paid to the study of speciation, yet many questions still remain. However, two new tools in the evolutionary biologist’s armory hold great promise. First, the ability to sequence entire genomes provides the opportunity to at last determine the exact changes involved in speciation. Second, the realization that evolutionary change can occur very rapidly, particularly when natural selection is strong, means that not only can evolution be observed in real time, but that it is even possible to conduct evolutionary experiments in nature. Synthesizing these two approaches provides unprecedented opportunity to unravel Darwin’s “mystery of mysteries.” We will focus on a model species of evolutionary studies, the brown anole lizard. Comparisons among populations will identify populations undergoing speciation and genome sequencing will examine its underlying genetic basis. Manipulative experiments on natural populations will then provide direct, real-time tests of hypotheses concerning the mechanisms of speciation. We will focus particularly on the question of evolutionary determinism: to what extent is evolution predictable? We will examine whether populations evolve similar adaptations when faced with similar environmental circumstances and, if so, whether they do so by the same genetic means. More generally, we will examine the extent to which speciation itself occurs deterministically, or whether progress toward reproductive isolation is idiosyncratic and non-repeatable from one speciation event to the next.