In dogs with neurological disturbances without myoclonus and extraneural signs, the clinical diagnosis of distemper is difficult perform. Considering the great infectious potential of the disease, the possibility of carrying out an antemortem diagnosis of distemper is important, particularly in hospitalized patients with neurological disease. The present study was carried out to evaluate RT-PCR for antemortem CDV detection in hospitalized dogs with neurological disturbances without the typical findings of distemper. We investigated five dogs with canine distemper virus (CDV) encephalomyelitis, in which the clinical diagnosis was not performed owing to the absence of characteristic signs of the disease, such as myoclonus and systemic signs. We observed an apparent high sensitivity of RT-PCR in urine samples for detection of CDV: four out of five urine samples were RT-PCR positive. The results of the present study suggest that urine is a good biological sample for antemortem CDV detection by RT-PCR in dogs with distemper encephalomyelitis in which the clinical diagnosis is likely to be difficult owing to the absence of suggestive distemper signs. The use of two different body fluids (urine and CSF) may increase the RT-PCR sensitivity for antemortem diagnosis of distemper in such cases.