The use of fish models has been proven to be an effective and sensitive tool for the evaluation of genotoxicity of pure compounds and complex mixtures of chemicals in the context of environmental screening of pollutants and hazard assessment in aquatic toxicology. In particular, fish cell lines have been successfully introduced for detection of genotoxic effects and can serve as an alternative to animal testing in preliminary eco-/genotoxicological studies. For this purpose comet assay has been extensively used in fish cell lines for the evaluation of genotoxic potential of chemicals and complex environmental matrices. The most often used fish cell lines in the comet assay are RTG-2, RTgill-W1 and RTL-W1 derived from rainbow trout (Onchorhynchus mykiss) gonads, gills and liver, respectively, and ZFL and ZF4 cells established from zebrafish (Danio rerio) liver and embryos, respectively. The present review gives an overview of the most often-used permanent fish cell lines in genotoxicology and discusses their application in the comet assay.