This study aimed to characterize the composition and distribution of the extracellular matrix (ECM) components in normal canine mitral valves (MV) and in chronic heart valve disease (CVD). MV of 50 dogs (normal (n=9), mild (n=13), moderate (n=17), severe (n=11) CVD) were investigated macroscopically, histologically (H.-E., picrosirius red) and immunohistochemically (collagen I, III, IV, V, VI, elastin, laminin, fibronectin, heparan sulphate). In normal MV, ECM components were expressed in a typical layered pattern. In mild CVD, basement membrane components (laminin, collagen IV, fibronectin) were increased. Advanced CVD was characterized by myxomatous nodular lesions displaying a marginal and a central region comprised mainly of collagen I, VI and fibronectin in the former and collagen I and III in the latter. Collagen IV and laminin appeared multifocally in marked CVD. In conclusion, not only an accumulation of proteoglycans, but also a distinctly altered expression of basement membrane components, and collagens characterizes CVD.