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The African black rhinoceros (Diceros bicornis) has adapted to a low iron diet during evolution and is thus prone to iron overload in captivity, which is associated with a number of serious disorders. A S88T polymorphism in the HFE gene has been suggested as a potential genetic basis of increased iron uptake in the black rhinoceros, while the Indian rhinoceros is thought to be unaffected by iron overload in captivity. In the present study, the histopathology and distribution of iron accumulations in five black rhinoceroses with iron overload syndrome were characterized and compared with three Indian rhinoceroses (Rhinoceros unicornis) and one African white rhinoceros (Ceratotherium simum). At necropsy examination, iron storage in black rhinoceroses was not associated with gross lesions. Microscopically, the most consistent and highest degree of iron load was found in the spleen, liver, small intestine and lung. There was minimal fibrosis and single cell necrosis in the liver. Endocrine organs, lymph nodes, heart and kidney were less often and less markedly affected. Unexpectedly, Indian rhinoceroses also showed iron load in the spleen and smaller amounts in organs similar to the black rhinoceros except for in the heart, while the white rhinoceros had only minor detectable iron storage in intestine, liver and lung. Sequence analysis confirmed the HFE S88T polymorphism in black but not in Indian rhinoceroses. The results indicate that Indian rhinoceroses may also be affected by iron storage in captivity, although in a milder form than the black rhinoceros, and therefore challenge the relevance of the S88T polymorphism in the HFE gene of black rhinoceroses as the underlying cause for iron overload.