Sourdough fermentations are characterized by the combined activity of lactic acid bacteria and yeasts. An investigation of the microbial composition of 21 artisan sourdoughs from 11 different Belgian bakeries yielded 127 yeast isolates. Also, 12 spontaneous 10-day laboratory sourdough fermentations with daily backslopping were performed with rye, wheat, and spelt flour, resulting in the isolation of 217 yeast colonies. The isolates were grouped according to PCR-fingerprints obtained with the primer M13. Representative isolates of each M13 fingerprint group were identified using the D1/D2 region of the large subunit rRNA gene, internal transcribed spacer sequences, and partial actin gene sequences, leading to the detection of six species. The dominant species in the bakery sourdoughs were Saccharomyces cerevisiae and Wickerhamomyces anomalus (formerly Pichia anomala), while the dominant species in the laboratory sourdough fermentations were W. anomalus and Candida glabrata. The presence of S. cerevisiae in the bakery sourdoughs might be due to contamination of the bakery environment with commercial bakers yeast, while the yeasts in the laboratory sourdoughs, which were carried out under aseptic conditions with flour as the only nonsterile component, could only have come from the flour used.