Ultraviolet B radiation is recommended for captive reptiles to stimulate production of adequate levels of vitamin D; however, little is known regarding the vitamin D status in many free-ranging populations. Current reference ranges for vitamin D in eastern box turtles have not yet been established. Sixty free-ranging eastern box turtles (Terrapene carolina carolina) from two well-studied populations in Illinois (n = 24) and Tennessee (n = 36) were assayed for plasma vitamin D concentration in 2014. There were no significant differences in concentrations between individuals in Illinois (mean: 117.5 nM/L) and Tennessee (mean: 98.7 nM/L) (P = 0.129) populations. Similarly, there were no differences in concentrations based on age class (P = 0.533) or sex (P = 0.532). There was a significant correlation between UV at the time of capture and vitamin D concentrations (R = 0.301, P = 0.030). Vitamin D was not correlated with total calcium (R = 0.018, P = 0.89) or Ca : P ratio (R = 0.025, P = 0.85). Diseases in captive individuals, including secondary nutritional hyperparathyroidism, may commonly be associated with vitamin D deficiencies, and clinical intervention relies on reference data. Vitamin D supplementation may be recommended if animals are deemed to be deficient. Data obtained can be used to improve the care of captive and free-ranging turtles by providing reference ranges, as well as better characterize the health of wild populations.