Contradiction exists on the incremental value of two-dimensional (2D) and 3D transoesophageal echocardiography (TOE) over 2D transthoracic echocardiography (TTE) for the detection of mitral valve (MV) prolapse in readers with different echocardiographic experience. Twenty patients and five healthy persons were retrospectively identified who had undergone 2D-TTE, 2D-TOE and 3D-TOE. Fifteen (75 %) patients had surgical evidence of prolapse of the posterior MV leaflet and five patients (25 %) had a dilated MV annulus without prolapse. Three reader groups with different echocardiographic expertise (novice, trainees, cardiologists) scored thus in total 675 posterior scallops. Overall there was an improvement in agreement and Kappa values from novice to trainees to cardiologists. Diagnostic accuracies of 2D-TOE were higher than those of 2D-TTE mainly in novice readers. The incremental value of 3D-TOE over 2D-TOE was mainly seen in specificities. Time to diagnosis was dramatically reduced from 2D to 3D-TEE in all reader groups (all P < 0.001). 3D-TOE also improved the agreement (+12 to +16 %) and Kappa values (+0.14 to +0.21) in all reader groups for the exact description of P2 prolapse. Differences between readers with variable experience in determining the precise localization and extent of the prolapsing posterior MV scallops exist in particular in 2D-TTE analysis. 3D-TOE analysis was extremely fast compared to the 2D analysis methods and showed the best diagnostic accuracy (mainly driven by specificity) with identification of P1 and P3 prolapse still improving from novice to trainees to cardiologists and provided optimal description of P2 prolapse extent.