Objective To assess systematically the accuracy of real-time three-dimensional transesophageal echocardiography (RT3D-TEE) in identifying involvement of mitral valve leaflets and scallops during mitral valvuloplasty. Methods One hundred and twenty-three patients with isolated mitral regurgitation for mitral valve repair were studied. Two-dimensional transesophageal echocardiography (2D-TEE) and RT3D-TEE were performed separately after general anesthesia. Two experienced interpreters analyzed the restored image offline and compared them with surgical findings. Results The diagnostic accuracy of RT3D-TEE in identifying mitral valve prolapse and its localization was significantly higher than that of 2D-TEE (P=0.001). There was no significant difference in diagnosing mitral valve chordal rupture between the two techniques (P=0.291), while the accuracy of RT3D-TEE in judging the region of chordal rupture was significantly higher (P<0.05). RT3D-TEE had a sensitivity of 100% in diagnosing simple leaflet cleft. Conclusion RT3D-TEE may accurately assess mitral valve prolapse, chordal rupture and its position, especially in diagnosing complex mitral valve prolapse and multiple chordal rupture. RT3D-TEE is thus a powerful tool in mitral valve repair, and is the most efficient modality in diagnosing mitral valve cleft.