Aims: The objective of this study was to determine the malachite green (MG) concentration in trout tissue and the effluent water of fish farms, at one of the largest trout fishery industries in Iran. Materiels and Methods: Twelve samples of water and fish tissue were collected from fish farms placed at the upstream ends of two large rivers in the study area. The samples, after extraction, were analyzed with liquid chromatography-mass spectrometry (LC-MS). The effluent water samples were also analyzed by the spectrophotometric method after cloud point extraction using the anionic surfactant Triton X-100. Results: The concentration of malachite green in the fish samples ranged from 265.2 to 1663 μg/kg, which is more than the recommended maximum allowable concentration by the Codex standards. MG in the water samples ranged from 5.65 ng/L to 384 μg/L. The equivalent concentrations of MG in the two large rivers in the study area were 1.78 and 0.62 ng/L, and the total MG load for these two rivers, with a fish production rate of 10,000 tons per year, was around 644 kg/d. Conclusion: We concluded that the concentration of MG used as an antimicrobial chemical in trout fish tissues and water samples in this study were out of compliance with the existing standards. Therefore, the fish products of these farms could cause serious public health hazards, and the discharge of the effluent from these farms, without treatment, posed potential environmental problems.