The comet assays or single cell gel electrophoresis is a simple, rapid, relatively inexpensive and very sensitive method that has been recently widely used to evaluate genomic integrity (or DNA damage) in aquatic animals exposed to diverse physical and chemical agents. However, the accuracy to evaluate comets assay has been the major obstacle in order to determine specific damage undergone cellular stress. The aim of this study was to evaluate DNA fragmentation in sperm cells based in fluorescence image analysis. The species used was the giant mussel Choromytilus chorus. The in vitro experiments were carried out through oxidative stress with five hydrogen peroxide concentrations (0, 50, 100, 500 and 1000 mM). To evaluate DNA damage two groups of parameters were used: (1) Morphological comet parameters as tail length, nuclear diameter (head) and tail moment, and (2) Quantification of DNA strand breaks grade by fluorescence image analysis. The sperm DNA damage was possitively correlated with the morphological parameters, as they were exposed to an increasing dose range of H2O2 and negatively correlated with nuclear diameter. Additionally, fluorescence analysis showed that fragment sizes and the fluorescence intensity could be identified and related the level of DNA damage independently the intercellular variation of DNA strand breaks. The large fragment sizes decrease with H2O2 doses, while medium and small fragments increase in treatments with highest peroxide concentration. This study supported future research to examine comet assay conjugated with molecular approaches, especially in reference to integrate FISH to evaluate if the DNA fragmentation includes specific genes.