Aims To investigate the Oenococcus oeni population occurring during spontaneous malolactic fermentation (MLF) of Amarone wine, a peculiar and hostile environment for malolactic bacteria. Methods and Results Pulsed-field gel electrophoresis (PFGE) analysis showed a high level of genetic heterogeneity within the O.oeni population involved in MLF throughout an industrial vinification of Amarone wine. The 13 strains with distinct PFGE profile displayed different capability to hydrolyse esters and glycosides, as well as great variability to growth under stress parameters, such as high ethanol content (15% v/v), low pH (3.0) and temperature (15 degrees C), and presence of SO2. Moreover, polymorphism in the gene sacB involved in exopolysaccharide production was observed among the strains. The strains showed differences to convert l-malic acid into l-lactic acid in wine. Conclusions The occurrence of spontaneous MLF in stressful ecosystems such as Amarone wine is related to the heterogeneity of O.oeni community; biodiversity indexes and strain evolution analyses suggested that its success depends on its initial strain evenness. Significance and Impact of the Study Remarkable intraspecies complexity within the O.oeni natural population could explain the great versatility of this species as key of successful adaptation to harsh winemaking conditions.