Progressive multifocal leukoencephalopathy (PML) is a rare but fatal demyelinating disease of the central nervous system (CNS) caused by JC virus (JCV). The current diagnostic standard for PML is real-time PCR testing of extracted DNA for assessing the presence of JCV DNA in cerebrospinal fluid (CSF). However, because of its sensitivity, real-time PCR assay for JCV testing has a risk of false-positive results due to DNA contamination. JCV isolates recovered from the brain or CSF of PML patients contain hypervariable mutations within the non-coding control region (NCCR) of the viral genome. In our laboratory, the high-resolution melting (HRM) assay was developed to distinguish the patient-dependent NCCR patterns of JCV DNA variants in clinical specimens. The HRM-based scanning of NCCR serves as a quick and convenient technique for comparing the mutational patterns of JCV variants in clinical samples and for the confirmation of PML diagnosis when combined with routine real-time PCR testing.