The DNA strand fragmentation as analyzed by the Comet assay is a common biomarker widely used to assess the genotoxic potential of chemical pollutants in marine organisms. However, basal levels of DNA strand breaks can be influenced also by factors unrelated to chemical exposure, and a better knowledge on the natural variability of such response would thus be important to discriminate the effects of anthropogenic activities. In this respect, the aim of the present work was to characterize the fluctuations of DNA strand breaks in mussels, Mytilus galloprovincialis, seasonally sampled over a 3 year period from a reference site along the Adriatic coast. Parameters reflecting DNA integrity exhibited significant seasonal changes in mussels haemocytes, with considerable differences between various sampling years. Comparing such results with those previously obtained on the natural fluctuations of trace metal concentrations and oxidative stress biomarkers in mussels tissues, significant correlations were obtained between DNA strand breaks with levels of Ba, Cd, Cu, Hg, Ni, Se, activity of catalase and content of malondialdehyde. Although mechanisms remain to be demonstrated, the overall results of this work suggest the potential role of natural prooxidant factors in affecting the baseline levels of DNA integrity in mussels, and confirm that natural variability of such responses should be carefully considered when monitoring the impact of genotoxins in marine ecosystems.