Background: Myxomatous mitral valve disease (MMVD) is more prevalent in Cavalier King Charles Spaniels (CKCSs) compared to dogs of other breeds at a given age. Abnormal valvular stress is thought to contribute to the development and progression of MMVD, and a relationship exists between mitral valve (MV) morphology and stress acting on the valve. Objectives: To determine whether the MV morphology of healthy adult CKCSs differs from the morphology of healthy adult dogs of other breeds determined by RT-3DTTE. Animals: Thirty-five healthy CKCSs and 41 healthy dogs of other breeds. Methods: Prospective cross-sectional study. Dogs underwent physical examination, conventional echocardiography, and RT-3DTTE. RT–3DTTE datasets were analyzed using dedicated software for MV morphologic analysis. Morphologic variables were compared between CKCSs and dogs of other breeds. Results: The MV of healthy CKCSs had a smaller annulus height (0.46 ± 0.11 vs. 0.56 ± 0.17; P =.0021), tenting height (0.26 ± 0.12 vs. 0.42 ± 0.18; P <.001), tenting area (0.42 ± 0.15 vs. 0.79 ± 0.34; P <.001), normalized tenting volume (0.09 [0.05–0.13] vs. 0.14 [0.10–0.20]; P <.001), and normalized area of the posterior leaflet (0.57 ± 0.15 vs. 0.66 ± 0.18; P =.016) compared to healthy dogs of other breeds; this results in CKCSs having a flatter MV with reduced tenting, compared to the MV of other breeds. Conclusions and Clinical Importance: These morphologic features could confer a mechanical disadvantage and play a role in the predisposition of CKCSs to the early development of MMVD.