Strope et al. present a new “100-genomes” strains resource for the model organism Saccharomyces cerevisiae. For a selected 93 strains, representing a diversity of geographical and environmental origins, the authors report whole-genome sequencing, de novo assembly and manual annotation to near-reference quality. Comparison of strains from human-associated environmental or clinical strains allowed the identification of genetic and phenotypic factors associated with clinical origins and the emergence of S. cerevisiae as an opportunistic pathogen.