Myxomatous mitral valve disease (MMVD) is the most common acquired cardiac disoder in dogs and a relevant model for human mitral valve disease. However, the molecular drivers of disease progression remain unclear, and reliable biomarkers for early diagnosis still hamper clinical management. This study investigated microRNA (miRNA) expression directly in histologically characterized mitral valve tissues. Formalin-fixed paraffin-embedded samples were obtained from control dogs (n = 7), low-grade MMVD (n = 8), and high-grade MMVD (n = 5). A bioinformatics workflow identified candidate miRNAs converging on extracellular matrix remodeling and canonical signaling pathways, including TGF-β, PI3K–Akt, and MAPK. Selected candidates, let-7 family, miR-98, miR-21, miR-30b, miR-133b, and miR-103, were validated by qPCR. Results revealed a general upregulation of the panel in MMVD compared with controls, with stage-dependent differences between low- and high-grade lesions. In particular, miR-21, let-7b, and miR-133b were markedly increased in advanced disease, while miR-30b emerged as an early-stage marker with potential prognostic value. These findings provide molecular evidence linking miRNA dysregulation to progressive valvular degeneration. By combining histologically defined tissue analysis with stage-based comparisons, this study identifies miRNAs with potential diagnostic and prognostic utility for canine MMVD.