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The involvement of DNA methylation in the response to cold stress of two different yeast species (<i>Naganishia antarctica</i>, psychrophilic, and <i>Naganishia albida</i>, psychrotolerant), exhibiting different temperature aptitudes, has been studied. Consecutive incubations at respective optimum temperatures, at 4 &#176;C (cold stress) and at optimum temperatures again, were performed. After Methylation Sensitive Amplified Polymorphism (MSAP) fingerprints a total of 550 and 423 clear and reproducible fragments were amplified from <i>N. antarctica</i> and <i>N. albida</i> strains, respectively. The two <i>Naganishia</i> strains showed a different response in terms of level of DNA methylation during cold stress and recovery from cold stress. The percentage of total methylated fragments in psychrophilic <i>N. antarctica</i> did not show any significant change. On the contrary, the methylation of psychrotolerant <i>N. albida</i> exhibited a nonsignificant increase during the incubation at 4 &#176;C and continued during the recovery step, showing a significant difference if compared with control condition, resembling an uncontrolled response to cold stress. A total of 12 polymorphic fragments were selected, cloned, and sequenced. Four fragments were associated to genes encoding for elongation factor G and for chitin synthase export chaperon. To the best of our knowledge, this is the first study on DNA methylation in the response to cold stress carried out by comparing a psychrophilic and a psychrotolerant yeast species.