The aim of the present study was the microbiological and technological characterization of laboratory- made sourdoughs for use in barley-flour-based bread-making. A defined multi-strain starter culture consisting of selected lactic acid bacteria (LAB) and yeasts from wheat sourdoughs was inoculated into three flour-water mixtures, composed of: (i) 100% wheat flour (ii) 50% wheat flour and 50% hull-less barley flour (composite flour); (iii) 100% hull-less barley flour. After two months of continuous propagation, the chemical characteristics of the three sourdoughs were investigated by measuring: pH, total titratable acidity and concentrations of various microbial metabolites by HPLC (i.e. lactic, acetic, phenyllactic and butyric acids and diacetyl). The microbial traits were studied through viable counts, isolation and typing of LAB and yeasts and PCR-DGGE analyses. Only Saccharomyces cerevisiae and Lactobacillus plantarum were detectable in the sourdoughs together with other lactobacilli species which were different depending on the type of flour blend used. The molecular typing of the isolates highlighted that only a few strains among those initially inoculated prevailed. The volume increases of the three types of sourdough were also investigated and a correlation was seen between an increase in the barley flour content and a reduction in the dough volume.