Three-dimensional transthoracic echocardiography (3D-TTE) provides a semi-automated proximal isovelocity surface area method (3D-PISA) to obtain quantitative parameters. Data assessing regurgitation severity in mitral valve prolapse (MVP) are scarce, so we assessed the 3D-PISA method compared with 2D-PISA and cardiovascular magnetic resonance (CMR) and the role of an eccentricity index. We evaluated the 3D-PISA method for assessing MR in 54 patients with MVP (57 ± 14 years; 42 men; 12 mild/mild-moderate; 12 moderate-severe; and 30 severe MR). Role of an asymmetric (i.e. eccentricity index ≥ 1.25) flow convergence region (FCR) and inter-modality consistency were then assessed. 3D-PISA derived regurgitant volume (RVol) showed a good correlation with 2D-PISA and CMR derived parameters (r = 0.86 and r = 0.81, respectively). The small mean differences with 2D-PISA derived RVol did not reach statistical significance in overall population (5.7 ± 23 ml, 95% CI − 0.6 to 12; p = 0.08) but differed in those with asymmetric 3D-FCR (n = 21; 2D-PISA: 72 ± 36 ml vs. 3D-PISA: 93 ± 47 ml; p = 0.001). RVol mean values were higher using PISA methods (CMR 57 ± 33 ml; 2D-PISA 73 ± 39 ml; and 3D-PISA 79 ± 45 ml) and an overestimation was observed when CMR was used as reference (2D-PISA vs. CMR: mean difference: 15.8 ml [95% CI 10–22, p < 0.001]; and 3D-PISA vs. CMR: 21.5 ml [95% CI 14–29, p < 0.001]). Intra- and inter-observer reliability was excellent (ICC 0.91–0.99), but with numerically lower coefficient of variation for 3D-PISA (8%–10% vs. 2D-PISA: 12%–16%). 3D-PISA method for assessing regurgitation in MVP may enable analogous evaluation compared to standard 2D-PISA, but with overestimation in case of asymmetric FCR or when CMR is used as reference method.