The comet assay is a sensitive technique to detect DNA damage caused by exposure to genotoxic chemical and physical agents and is widely used in ecotoxicology. The assay has been applied in aquatic species, mainly fish and bivalves, in field biomonitoring programs and in experimental studies. The aim of the present study was to retrieve and review the published evidence to define the role of the comet assay in the assessment of genotoxic pollutants. The study focused on the application of the test in Mytilus sp, used as a sentinel organism. Twenty-one biomonitoring studies, carried out in wild and in transplanted mussels, were evaluated. An increase of the comet parameters in animals from polluted areas with respect to the controls was observed in the majority of the studies with a large variability (frequency ratio:1.2–14.5) associated with types and extent of exposure to pollutants. Three studies out of 21 reported a lack of response. Heavy metals, polycyclic aromatic hydrocarbons (PAH), polychlorinated biphenyls (PCB) and pesticides were the main types of chemicals detected in sediments and/or mussel tissues from polluted areas. Twenty-nine laboratory studies were retrieved showing the sensitivity of the comet assay in detecting DNA damage in mussels exposed to the most relevant pollutants and mixtures of relevant contaminants, such as pharmaceuticals, anti-fouling agents or crude oil. The comet test also appeared to be a suitable approach to detect the genotoxic effects of nanoparticles. In some studies problems in the interpretation of data or discrepancies between the results from different laboratories were noted. Critical steps in experimental protocol and characterization of pollution, environmental variables such as temperature, salinity, food availability, physiological and pathological status of the animals are important factors which should be controlled and considered in the analysis of the results.