The Saccharomycetales or ‘true yeasts’ consist of more than 800 described species, including many of scientific, medical and commercial importance. Considerable progress has been made in determining the phylogenetic relationships of these species, largely based on rDNA sequences, but many nodes for early-diverging lineages cannot be resolved with rDNA alone. rDNA is also not ideal for delineating recently diverged species. From published full-genome sequence data, we have identified 14 regions of protein-coding genes that can be PCR-amplified in a large proportion of a diverse collection of 25 yeast species using degenerate primers. Phylogenetic analysis of the sequences thus obtained reveals a well-resolved phylogeny of the Saccharomycetales with many branches having high bootstrap support. Analysis of published sequences from the Saccharomyces paradoxus species complex shows that these protein-coding gene fragments are also informative about genealogical relationships amongst closely related strains. Our set of protein-coding gene fragments is therefore suitable for analysing both ancient and recent evolutionary relationships amongst yeasts.