The authors have developed a stentless, chordally supported quadricusp mitral bioprosthesis made from bovine pericardium. A large anterior, two commissural, and one posterior cusp all have straight hinge lines and avoid the excessive flexion stress of stent supported valves. The chordal support is concentrated in two flaps for suture to the papillary muscles. Previous in vitro and animal studies demonstrated its superior hemodynamic, biomechanical, and biocompatible behavior over commercial valve bioprostheses. In this study, we further evaluated its in vivo performance by intraoperative epicardial Echo/Doppler immediately after the bioprosthesis was implanted in 10 sheep. In all 10 sheep, Echo showed the valves were similar to native mitral valves in both anatomic appearance and movement. Mitral annular motion and annular-papillary muscle integrity were preserved. In systole, the valves closed competently with chordae moving upward and straightening; in diastole, the valves opened wide with chordae moving downward and relaxing. Color and pulsed Doppler showed no signs of stenosis or regurgitation. In conclusion, the new mitral bioprosthesis provide excellent in vivo performance that mimics the native mitral valve. Intraoperative echocardiography is recommended after mitral valve replacement with such valves.