The aim of this study was to define reference values for vertebral heart score (VHS) and modified left atrium (LA)-VHS, cardiac structures, and major thoracic vessels measurements and ratios obtained from thoracic contrast radiography in Wistar albino rats. VHS, modified LA-VHS, left (LV) and right (RV) ventricles, interventricular septum (IVS), aorta (Ao), caudal vena cava (CaVC), and fourth thoracic vertebrae (v) length (T4) were measured from left lateral thoracic contrast radiographs of 50 young, healthy adult male Wistar albino rats. LV/T4, RV/T4, IVS/T4, Ao/T4, CaVC/T4, and CaVC/ Ao ratios were calculated from these values. Measurements were performed by two observers unaware of the signalment data. Median values and references ranges were 8.2v (7.4-9.1) for VHS, 1.2 (1.0-1.5) for modified LA-VHS, 7.8 mm (6.2-9.1) for LV, 3.4 mm (2.8-4.5) for RV, 2.1 mm (1.8-3.0) for IVS, 2.1 mm (1.8-2.8) for Ao, 2.2 mm (1.7-3.0) for CaVC, 4 mm (3.5-4.5) for T4, 2 (1.5-2.3) for LV/T4, 0.85 (0.68-1.22) for RV/T4, 0.52 (0.42-0.83) for IVS/T4, 0.53 (0.42-0.75) for Ao/T4, 0.55 (0.45-0.7) for CaVC/T4 and 1.05 (0.74-1.1.37) for CaVC/Ao. Further studies are now needed to determine whether measurement values obtained from contrast radiography in rats are useful in the diagnosis of cardiomyopathy and heart failure. The radiographic measurement values presented in this study can be used as a reference baseline for both pet and laboratory rats.