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OBJECTIVE To determine the prevalence of nucleic acid from selected cardiotropic pathogens in endomyocardial biopsy samples from dogs with unexplained myocardial and rhythm disorders (UMRD) and compare prevalence with that for a group of control dogs with congenital heart disease (CHD). ANIMALS 47 client-owned dogs. PROCEDURES Right ventricular endomyocardial biopsy was performed in dogs with UMRD (dilated cardiomyopathy [n = 25], atrioventricular block [6], and nonfamilial ventricular [4] and supraventricular arrhythmias [2]) or CHD (10) that required right ventricular catheterization. Biopsy samples were evaluated histologically, and PCR assays were used for detection of nucleic acid from 12 pathogens. RESULTS 197 biopsy samples were collected from dogs with UMRD (n = 172) or CHD (25). At least 1 pathogen was detected in 21 of 37 (57%; 95% confidence interval [CI], 41% to 71%) dogs with UMRD, and canine coronavirus was detected in 1 of 10 (10%; 95% CI, 2% to 40%) dogs with CHD. Dogs with UMRD were significantly more likely than dogs with CHD to have pathogens detected in biopsy samples (OR, 11.8; 95% CI, 1.3 to 103.0). The most common pathogens in dogs with UMRD were canine distemper virus, canine coronavirus, canine parvovirus 2, and Bartonella spp. No pathogens were detected in available blood samples from dogs with pathogens detected in biopsy samples. CONCLUSIONS AND CLINICAL RELEVANCE Detection of nucleic acids from selected cardiotropic pathogens in myocardial tissue from dogs with UMRD suggested a possible association between the 2. Further studies are needed to explore whether this association is causative or clinically important. (J Am Vet Med Assoc 2019;255:1150-1160).