Oenococcus oeni strains from traditional Italian red wines of the Basilicata region were investigated on the basis of their physiological and molecular response to different temperatures and ethanol concentrations. All strains were highly resistant to different ethanol concentrations and it has been observed that 7% ethanol was able to stimulate the growth of strains in wine, and 12–13% of ethanol allowed their proliferation. Moreover, strain tolerances to 18 and 42 °C were observed. Fingerprinting analysis with fluorescent differential display-PCR and investigation of changes in gene expression during the tolerance process were carried out. The expression gene pattern reflects mechanisms involved in tolerance to environmental conditions. This study establishes and validates a method that enables, with a high reproducibility, different gene expression identification under stress conditions in lactic acid bacteria.