Mitral valvular prolapse (MVP) in dogs is characterized by myxomatous valvular degeneration, which is caused by abnormal valvular thickening and incomplete coaptation of the mitral valve leading to mitral regurgitation. Mitral regurgitation causes left atrial and left ventricular enlargement. Pathogenesis of the disease is unknown, although some studies have suggested the involvement of endothelin and systemic connective tissue diseases. Mitral valvular prolapse in dogs commonly occurs in aged small dog breeds, including Malteses and Shih Zhus. This case study investigated the clinical features of an affected Maltese family and performed pedigree analysis. To the best of the authors’ knowledge, this is the first report of putative familial mitral valve prolapse and regurgitation in Maltese dogs. All family members in this study showed degenerative valvular changes and echocardiographic features of mitral valvular prolapse. Although disease progression differed, all dogs progressed to advanced heart failure stage within 2-3 years after diagnosis. Therefore, this is the first study to identify putative familial mitral valve prolapse in Maltese dogs. This finding suggests strong genetic etiology involved in the development of degenerative mitral valve disease in Maltese dogs. Furthermore, this finding could be a valuable resource for the identification of gene mutations in dogs with familial mitral valvular prolapse.