In the present study, a total of eight sourdough samples were collected from three different bakeries at two different times in Turkey. Also, laboratory-scale sourdough production was conducted by daily back-slopping for 7 days. Microbiological and chemical properties of the sourdoughs were investigated. Yeast species in the sourdoughs were identified by subjecting all presumptive yeast cultures to internal transcribed spacer region amplification of the 5.8S rRNA gene, restriction fragment length polymorphism analysis usingHae III,Hha I, andHinf Iendonucleases, and sequence analysis of the D1/D2 domain of the 26S rDNA gene. A total of seven profiles were determined according to the restriction fragments. Totally, 148 yeast isolates were identified at the species level (>= 400bp, 99% identity) asSaccharomyces cerevisiae(106),Kazachstania bulderi(11),Pichia fermentans(nine),Pichia membranifaciens(eight),Kazachstania servazzii(seven),Kazachstania unispora(four), andHanseniaspora valbyensis(three). Although collected sourdoughs were produced without using baker's yeast,S.cerevisiaewas the most frequently isolated yeast species. This can be related to the contamination of the bakery environment with commercial baker's yeast during the production of other bakery products. The pH and acidity levels of the collected sourdough samples ranged from 3.71 to 3.96 and 6.78 to 23.93 mL 0.1 N NaOH/10 g dough, respectively. Mean values of the content of maltose + sucrose, glucose, fructose, lactic acid, and acetic acid were 2.43, 1.57, 2.67, 7.30, and 1.40 g/kg, respectively. Due to the artisan and region-dependent handling of the sourdough, different biochemical patterns were observed among the collected samples.