Vertebral heart scoring (VHS) is a semiquantitative method to assess the presence and severity of cardiomegaly by usingthoracic radiographs. VHS in rhesus macaques (Macaca mulatta) has not been validated or used routinely in the clinical orresearch setting. We hypothesized that rhesus macaques with cardiac disease diagnosed by using echocardiography wouldhave higher VHS than animals without cardiac disease. A total of 150 rhesus macaques were enrolled in this study. All animalsunderwent echocardiography and thoracic radiography (right lateral [RL], dorsoventral [DV], and ventrodorsal [VD] views).According to echocardiography, 121 rhesus macaques had no cardiac disease and were used to establish reference intervalsfor VHS. The remaining 29 macaques had hypertrophic cardiomyopathy (n = 20) or other cardiac disease (n = 9). Resultsshowed that VHS of RL and VD views were significantly higher in macaques with any of the identified cardiac diseases andin the cardiac disease group that excluded hypertrophic cardiomyopathy. VHS of animals with HCM was not significantlydifferent than that of control animals. In the RL view, VHS was moderately accurate for predicting the presence of cardiacdisease, with an AUC of 0.71 and an optimal cut-off value of 10.25 (sensitivity: 62%, specificity: 77%). In the VD view, VHSwas a mildly accurate test for cardiac disease, with an AUC of 0.654 and an optimal cut-off value of 10.65 (sensitivity, 66%;specificity, 63%). Study results indicated that VHS could be a useful screening tool for clinically identifying rhesus macaqueswith cardiac disease. However, VHS is unlikely to replace echocardiographic examination for determining the presence, type,and severity of cardiac disease in this species.