Aim: To characterize the genetic and phenotypic diversity of 33 strains of Lactobacillus rossiae. Methods and Results: Genotypic identification was carried out by partial 16S rRNA gene sequence analysis. Genetic diversity was evaluated by RAPD–PCR analysis. Phenotypic diversity was evaluated through fermentative profile by Biolog system, proteinase and peptidase activities using synthetic substrates, and acidification capacity and amino acid profile during sourdough fermentation. The genetic analyses excluded clonal relatedness among the strains used. A large phenotypic diversity was found. It mainly concerned the capacity to use carbon sources available in sourdough during fermentation, the quotient of fermentation and the peptidase activities, especially towards proline containing synthetic substrates. The free amino acid profiles differed either for the total concentration or for the type of amino acids. With a few exceptions, proteinase activity towards wheat albumins and globulins was weak. Conclusions: Overall, no relationships between genetic and physiological analyses were found, and the strains examined showed a marked genetic and phenotypic heterogeneity. L. rossiae strains had interesting properties for application in sourdough fermentation. Although some strains combined several technological traits, the association of more strains seemed to be a requisite to get optimal sourdough characteristics. Significance and Impact of the Study: It represents the first characterization of the diversity within the L. rossiae species. Besides, it may represent an example of computerized analysis of genotypic and phenotypic information to select strains for improving sourdough characteristics.