Hypertrophic cardiomyopathy (HCM) is the most common heart disease in cats, characterized by primary left ventricular hypertrophy. Feline HCM closely resembles human HCM and is suggested as translational animal model for the human disease. A genetic cause is established in humans and suspected for cats, but little is known about the gene expression and pathways involved in the pathogenesis of HCM. To investigate the myocardial transcriptome changes in HCM, RNA sequencing was conducted on left ventricle (LV) and left atrium (LA) samples of healthy cats and cats with HCM (each n = 5; 20 samples). Ingenuity Pathway Analysis was used to determine functional pathways, regulators, and networks. Distinct gene expression profiles were identified in the LV and LA of the feline healthy and HCM myocardium. Analysis of differentially expressed mRNAs (>2 fold; FDR < 0.01) found chamber-specific (LV vs. LA) expression in both healthy and HCM groups, with higher transcriptional activity in the LA. Genes that contribute to the distinct structure and function of each chamber in health and HCM were identified in the regional comparison. The gene expression profiles of HCM compared to healthy hearts revealed disease related genes, including THBS4 and KLHL33 (LV), FAM177B and THRSP (LA), the latter 3 have not been reported for the myocardium so far, as the top differently expressed genes in the HCM heart. Differently expressed genes and functional pathways found in the HCM heart are associated with cardiac remodeling and fibrosis, inflammation, microvascular changes, calcium signaling and cardiac metabolism, with some regional differences. RhoGDI-RhoGTPase signaling, integrin and ILK signaling pathways, the LXR/RXR pathway in the LA, and the PPARα/RXRα, HIF1α and CXCR4 pathways in the LV might be of particular importance in the HCM disease process. This study identified region-specific myocardial gene transcription patterns as well as novel genes and pathways associated with HCM.