Aflatoxin B1 (AFB1) is a mycotoxin that causes cytotoxicity through oxidative damage to its target organs. The liver is the first target of AFB1 damage. The aim of this study was to evaluate the protective effect of selenium on AFB1-induced hepatic mitochondrial damage in ducklings using molecular biological and histopathological techniques. Aflatoxin was administered via intragastric intubation (0.1 mg/kg body weight), daily for 21 days. The experimental group also received intragastric sodium selenite (1 mg/kg body weight), while the control group was given the same volume of dimethyl sulfoxide (DMSO). Sequence analysis of the mitochondrial DNA D-loop region showed that AFB1 induced damage. All AFB1-administrated ducklings were identified as having D-loop mitochondrial DNA mutations. Mutations were detected in two ducklings that had received both AFB1 and selenium. Mitochondrial swelling assays showed that opening of the mitochondrial permeability transition pores was increased in ducklings that had received AFB1 for 14 and 21 days (P < 0.05). Selenium significantly attenuated these adverse effects of AFB1. After AFB1 exposure, histological alterations were observed, including fat necrosis, steatosis, and formation of lymphoid nodules with infiltrated lymphocytes. These histological abnormalities were also attenuated by treatment with selenium. The overall data indicated that selenium exerts a potent protective effect against AFB1-induced hepatic mitochondrial damage, possibly through its antioxidant activity.