Color M-mode echocardiography has recently been utilized to describe diastolic flow propagation velocity (Vp) in the left ventricle. While increasing temporal resolution from 15 to 200 Hz, this M-mode technique requires the user to select a single scanline, potentially limiting quantification of Vp due to the complex three-dimensional inflow pattern. We previously performed computational fluid dynamics simulations to demonstrate the insignificance of the scanline orientation, however geometric complexity was limited. The purpose of this study was to utilize high frame-rate 2D color Doppler images to investigate the importance of scanline selection in patients for the quantification of Vp. 2D color Doppler images were digitally acquired at 50 frames/s in 6 subjects from the apical 4-chamber window (System 5, GE/Vingmed, Milwaukee, WI). Vp was determined for a set of scanlines positioned through 5 locations across the mitral annulus (from the anterior to posterior mitral annulus). An analysis of variance was performed to examine the differences in Vp as a function of scanline position. Vp was not effected by scanline position in sampled locations from the center of the mitral valve towards the posterior annulus. Although not statistically significant, there was a trend to slower propagation velocities on the anterior side of the valve (60.8 +/- 16.7 vs. 54.4 +/- 13.6 cm/s). This study clinically validates our previous numerical experiment showing that Vp is insensitive to small perturbations of the scanline through the mitral valve. However, further investigation is necessary to examine the impact of ventricular geometry in pathologies including dilated cardiomyopathy.