Captive cheetahs often demonstrate a high incidence of diseases in which vitamin A imbalances are implicated. These can occur even under controlled and optimised feeding regimens, which is why surveillance of vitamin A status is mandatory in the successful health management of cheetahs. Serum levels of the vitamin do not reflect the true vitamin A status and liver tissue analysis is rather impractical for routine application in large felids. A biomarker for evaluating overt and subclinical vitamin A deficiency in cheetahs is needed. This study evaluates whether increased calvarial bone thickness can be detected on routine skull radiographs of vitamin A deficient cheetahs compared to unaffected animals, and secondly, evaluates whether there is increased bone thickness in clinically sound captive cheetahs in general compared to wild-living controls. Bone thickness in the neuro- and splanchnocranium was measured in 138 skull radiographs. Significant thickening of the parietal bones was found in latero-lateral radiographs of immature cheetahs (< 12 months) with vitamin A deficiency. This finding may allow a presumptive diagnosis of hypovitaminosis A in immature cheetahs. A general difference in skull thickness between free-living and captive cheetahs was not found.