The objective of this study was to determine the intraoperator, intraobserver, and interobserver repeatability in a series of conventional echocardiographic parameters and in some of the newer measurements of diastolic function, including color M-mode flow propagation velocity, isovolumic relaxation time and pulsed-wave Doppler tissue imaging velocities. Four healthy cats were each scanned five times over a 3-day period. The repeatability of these echocardiographic analyses was compared using Bland–Altman analysis (intraoperator repeatability). After a minimum of 5 weeks, one scan was randomly selected from each cat, and was remeasured by the original observer and the results compared using a standard paired Student's t-test (intraobserver repeatability). One scan from each cat was then randomly selected and two observers, with similar levels of experience, measured each of these scans. The repeatability of these echocardiographic analyses was compared using Bland–Altman analysis (interobserver repeatability). The conventional two-dimensional (2D), M-mode and spectral Doppler measurements were repeatable in both their acquisition and measurement by a single investigator; there was a greater degree of variation between the two observers. The predominant (S′, E′, and A′) pulsed-wave Doppler tissue imaging velocities from the left apical four-chambered view, generally had a coefficient of variation of approximately 20% (range 9.62–34.08%). However, with pulsed-wave Doppler tissue imaging, velocities recorded during the isovolumic phases, the velocity of the tricuspid annulus, and the radial fiber velocity within the interventricular septum, frequently had coefficients of variation in excess of 20% and should therefore be interpreted with caution.