Allozyme electrophoresis was used to characterize 39 isolates belonging to the wild yeast species Saccharo- myces paradoxus for variation at nine enzyme loci. The data revealed significant genetic differentiation between isolates from two geographically distinct regions, one including continental Europe and the other including the Russian Far East and Japan. The results are consistent with previous observations indicating that there is partial reproductive isolation between isolates collected from these regions, and they suggest the possibility that these two populations represent an early stage in speciation. The evolutionary genetics of yeasts has received little atten- tion compared with the classical and molecular genetics of these organisms, especially as developed in Saccharomyces cer- evisiae. Indeed, S. cerevisiae is widely regarded as a domesti- cated organism and is seldom collected away from sources related to human activity, so that studies of wild S. cerevisiae populations are problematical. However, at least six sibling species have been identified in the Saccharomyces sensu stricto complex (S. cerevisiae, Saccharomyces paradoxus, Saccharomy- ces bayanus Hansen, one Brazilian Saccharomyces sp., and two Japanese Saccharomyces spp.) by genetic analysis (15, 18) and DNA-DNA reassociation studies (7, 33-35, 38), and some of these species appear to exist only in the wild state; that is, they are not found in any human baking or fermenting applications. Significantly, classification in the Saccharomyces sensu stricto complex is in accord with the biological species concept (12); interspecific matings yield viable hybrids, but these hybrids produce nonviable ascospores and are consequently sterile. In contrast, the products of intraspecific matings produce highly viable ascospores (13-15, 21, 23). The existence of interfertile populations of wild Saccharomyces yeasts opens the way to studies of the evolutionary systematics and population genetics of yeasts similar to studies conducted with higher eukaryotes and various other fungi (27, 28, 36, 37). In this paper, we describe a study of genetic variation in wild populations of S. paradoxus. S. paradoxus Batchinskaya (synonyms, S. cerevisiae var. tet- rasporus (Beijerinck ex Dekker) Phaff et al., S. cerevisiae var. terrestris Jensen, and Saccharomyces douglasii nom. nud. ( 14, 191) is the closest relative of S. cerevisiae Hansen. The level of total DNA homology (as determined by reassociation studies) between S. cerevisiae and S. paradoxus is approximately 50%. (Some recent studies found considerably higher levels of ho- mology between DNA sequences from individual genetic loci (l, 111). The levels of total DNA homology between S. cerevi- siae and the other sibling species are lower (7, 33-35, 38). While performing genetic identification studies of S. paradoxus isolates collected in various regions of the world, we noticed that isolates collected in Europe formed highly fertile hybrids with a reference tester strain of S. paradoxus from Denmark, whereas isolates collected from other regions, particularly from the Russian Far East and Japan (referred to as Far East