An important step in predicting the effects of future increases in UV radiation (UVR) is to evaluate the mechanisms that organisms use to prevent and repair DNA damage and determine how those mechanisms influence UVR sensitivity. Damage is prevented to varying degrees through photo-protection and repaired via two main pathways: nucleotide excision repair and photoenzymatic repair. At present, little is known about the generality or similarity of these defenses among temperate freshwater fishes. We used laboratory experiments to compare UVR defense mechanisms among five freshwater fish species representing four families and three orders. Purified DNA, freeze-killed larvae and live larvae were exposed to UVB radiation for 12 h in the presence or absence of photorepair radiation. After exposure, we quantified frequencies of cyclobutane pyrimidine dimers in each exposure treatment. All five species used photoprotection and proportional decreases in dimer frequency were similar for all species. Evidence of excision repair was also found for all species but proportional decreases in photoproduct frequencies varied among species. Finally, evidence of photoenzymatic repair was found for only two of the five species.