Three-dimensional echocardiography (3DE) is advised for right ventricular (RV) assessment. Data regarding the optimal acquisition settings and optimization are still scarce. We aimed to evaluate the feasibility, reproducibility and validation of 3DE for RV volume and function assessment, using cardiac magnetic resonance (CMR) as gold standard. Thirty healthy volunteers and 36 consecutive patients were prospectively included. CMR was performed in the latter. Standard apical four-chamber view (A4CV), focused A4CV and modified A4CV were used for 3DE RV acquisition. Feasibility (and the effect of changes in settings) was evaluated. Intra and interobserver analyses were performed by three observers (expert vs. novice). RV parameters by echocardiography were compared to CMR. Feasibility of acquisition was 16.7% for A4CV, 80.0% for focused A4CV and 16.7% for modified A4CV. Changes in settings had no significant influence on feasibility and further analysis. Intraobserver variability was good in both expert and novice, interobserver variability was good between experienced observers. Compared to CMR, 3DE volumes were significantly lower with fair to moderate correlation (EDV: 91.1 ± 24.4 mL vs. 144.3 ± 43.0 mL (p < 0.001), r = 0.653 and ESV: 48.1 ± 16.4 mL vs. 60.4 ± 21.2 mL (p < 0.001), r = 0.530, by multi-beat 3DE and CMR respectively). These findings suggest that standardization is needed in order to implement this technique in clinical practice, thus further studies are required.