Aims: To characterize the genetic and phenotypic diversity of 135 lactic acid bacteria (LAB) strains isolated from Italian wines that undergone spontaneous malolactic fermentation (MLF) and propose a multiphasic selection of new Oenococcus oeni malolactic starters. Methods and Results: One hundred and thirty-five LAB strains were isolated from 12 different wines. On the basis of 16S amplified ribosomal DNA restriction analysis (ARDRA) with three restriction enzymes and 16S rRNA gene sequencing, 120 O. oeni strains were identified. M13-based RAPD analysis was employed to investigate the molecular diversity of O. oeni population. Technological properties of different O. oeni genotypes were evaluated in synthetic medium at increasing selective pressure, such as low pH (3 center dot 5, 3 center dot 2 and 3 center dot 0) and high ethanol values (10, 11 and 13% v/v). Finally, the malolactic activity of one selected strain was assessed in wine by malolactic trial in winery. Conclusions: The research explores the genomic diversity of wine bacteria in Italian wines and characterizes their malolactic metabolism, providing an efficient strategy to select O. oeni strains with desirable malolactic performances and able to survive in conditions simulating the harsh wine environment. Significance and Impact of the Study: This article contributes to a better understanding of microbial diversity of O. oeni population in Italian wines and reports a framework to select new potentially O. oeni starters from Italian wines during MLF.