This study aimed to investigate effects of malathion on DNA breakage in the liver and gill of rainbow trout (Oncorhynchus mykiss) in the laboratory condition. The experiment was performed with four concentrations of malathion, i.e. 0 (control), 0.025, 0.05 and 0.075 mg L‑1 and three replicate for nine days. The liver and gill were sampled on the first, fifth and ninth days after the start of the experiment and stored in microtubes containing ethanol. The DNA was extracted using a kit (Cinnapure), electrophoresed and photographed using the gel doc system. The percent of DNA breakage was determined using fuzzy logic and the weighted average. Malathion concentration and time had significant effects on the DNA breakage. Also there was a significant interaction between malathion concentration and time on the DNA breakage. The greatest damage in the liver was found in specimens exposed to 0.075 mg L‑1 and for the gill, in those exposed to 0.05 mg L-1 nine days after the start of the experiment indicating a lack of a mechanism in both tissues to repair the damage malathion. The highest level of DNA damage was found in the liver. Thus, DNA breakage can be used as an appropriate method for detecting genetic damage of environmental pollution that may serve as a biological marker in ecotoxicological studies.