ABSTRACT Here we report the isolation of four Saccharomyces species (former Saccharomyces sensu stricto group) from tree bark. The employment of two temperatures (10°C in addition to the more commonly used 30°C) resulted in the isolation of S. kudriavzevii and S. uvarum , two species that grow at low temperatures, in addition to S. cerevisiae and S. paradoxus . A clear bias was found toward the bark of certain trees, particularly certain oak species. Very often, more than one Saccharomyces species was found in one locality and occasionally even in the same bark sample. Our evidence strongly suggests that (markedly) different growth temperature preferences play a fundamental role in the sympatric associations of Saccharomyces species uncovered in this survey. S. kudriavzevii was isolated at most of the sites sampled in Portugal, indicating that the geographic distribution of this species is wider than the distribution assumed thus far. However, the Portuguese S. kudriavzevii population exhibited important genetic differences compared to the type strain of the species that represents a Japanese population. In this study, S. kudriavzevii stands out as the species that copes better with low temperatures.