The genetic diversity of 339 strains of lactic acid bacteria responsible for the spontaneous malolactic fermentation (MLF) in Tempranillo wines produced in six cellars located in different provinces of Castilla-La Mancha region of Spain was analyzed by using randomly amplified polymorphic DNA (RAPD). The results indicate that Oenococcus oeni was the predominant species in MLF of Tempranillo wines with a scant diversity of other lactic acid bacteria (LAB) species. The presence of a well-adapted cellar microbiota is confirmed, with MLF dominated by a limited number of O. oeni genotypes. Clustering of isolates from samples taken from different provinces showed the existence of coincident genotypes in different cellars of the region. This result suggests that a population of cosmopolitan O. oeni strains exists in the Castilla-La Mancha wine-growing region. (C) 2009, The Society for Biotechnology, Japan. All rights reserved.