Successful mitral valve (MV) repair for degenerative mitral regurgitation (DMR) is mainly related to surgical expertise and MV anatomy. Although 2D echocardiography, specifically transoesophageal (TOE), provides precise information regarding MV anatomy, recent advancements in matrix technology meant a decisive step forward to the point where segmental MV analysis can be accurately performed from a noninvasive 3D transthoracic (TTE) approach. The aims of this study were: (a) to evaluate the feasibility and time required for real-time 3D TTE in a large consecutive cohort of patients with severe DMR in the assessment of MV anatomy; (b) to compare the accuracy of 3D TTE and 2D TOE versus surgical inspection in the recognition and localization of all components of the MV leaflets; (c) to establish the added diagnostic value of 3D colourDoppler examination to pure 3D morphologic evaluation. 149 consecutive patients with severe DMR underwent complete 3D TTE before surgery and 2D TOE in the operating room. Echocardiographic data obtained by the different techniques were compared with surgical inspection. 3D TTE was feasible in a relatively short time (8 ± 4 min), with good (49%) and optimal (33%) imaging quality in the majority of cases. 3D TTE had significant better overall accuracy compared to 2D TOE (93 and 91%, p < 0.05, respectively). 2D TOE was significantly more specific than 3D TTE in the identification of A3 prolapse (99 vs. 96%). The colourDoppler mode did not improve significantly the accuracy of 3D TTE, albeit it determined a better sensitivity in the detection of A2 prolapse if compared to 2D TOE (95 vs. 85%). 3D TTE with or without colourDoppler is a feasible and useful method in the analysis of MV prolapse; it allows a preoperative and noninvasive description of the pathology as accurate as the 2D TOE.