Vitamin E and selenium (Se) are important in animal health and work in different ways as antioxidants. Se, as a micronutrient, can also cause toxicity if ingested in high amounts. Steatitis due to possible vitamin E and/or Se deficiency has been described in reptiles in several cases while toxicity associated with high levels of Se has been described in turtles. While environmental and dietary factors are known to contribute to vitamin E and Se levels in animal tissues, there is a lack of information on expected levels of both substances in blood of various reptile species as a baseline for evaluation of health issues due to under- or over provision in the diet. We measured vitamin E and Se in heparinized plasma samples from Hermann´s tortoises (Testudo hermanni) (n = 522) and red-eared sliders (Trachemys scripta elegans) (n = 188) from different regions in Germany over the course of one year. Levels of both differed significantly (p < 0.05) depending on species, season, and region and vitamin E levels differed depending on sex. Both Se and vitamin E levels were higher in the sliders than in the tortoises and Se varied more depending on the region in the sliders. The different diets and habitats of the two species probably play a major role in expected blood levels of both substances. This work provides a baseline for understanding the role of various factors in vitamin E and Se metabolism as well as the role of these substances in disease in chelonians.