The B-complex vitamins play an important role in many metabolic processes in the body, and deficiencies in individual B vitamins can have serious health consequences, e.g. neurological disorders as a consequence of vitamin B1 deficiency. However, this group of substances has not yet been studied extensively in reptiles and reference intervals, for example from the blood, are completely lacking. The aim of this study was therefore to measure the vitamins B1 (thiamine pyrophosphate), B2 (flavin adenine dinucleotide), B6 (pyridoxal), B9 (folic acid), and B12 (cobalamin) in the blood of healthy adult slider turtles (Trachemys scripta) and to establish suitable reference intervals for this species. Heparin whole blood and plasma from 165 clinically healthy slider turtles under managed care from different keepers in Germany were analyzed using high performance liquid chromatography (HPLC) and spectrometry. The following median values were determined: vitamin B1 59.10 µg/L; B2 693.50 µg/L; B6 2,37 µg/L; B12 3225.00 pg/mL; folic acid 16.06 ng/mL. The results also showed that the values for vitamins B1 and B9 in the blood varied significantly (p < 0.0001) depending on the season. This study provides an important basis for further investigations, for example in optimizing diets and determining the role of inadequate provision of dietary B-complex vitamins in clinical disease.