JC polyomavirus (JCV) is the causative agent of progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease. JCV isolates from PML patients have hypervariable mutations in the noncoding control region (NCCR) of the viral genome. Although nucleotide sequencing analysis of NCCR mutation is useful for the confirmation of PML diagnosis and basic studies examining JCV variants, it is often labor-intensive, time-consuming, and expensive. This study was conducted to evaluate the feasibility of a high-resolution melting (HRM) analysis technique for the rapid and low-cost scanning of NCCR mutations. The real-time PCR-HRM assay was developed with a pair of primers targeting the NCCR, and mutational patterns of NCCRs were compared using sequence-confirmed JCV DNA clones and CSF DNAs from PML patients. The NCCR patterns of DNA clones of the archetype JCV and PML-type variants could be differentiated by PCR-HRM. The mutational patterns of the rearranged NCCR clones were similar to those of JCV variants in the original CSF specimens as judged by nested PCR-HRM using pre-amplified targets. In addition, nested PCR-HRM could distinguish NCCR mutations in the JCV DNAs from each specimen at the patient level. These results indicate that the HRM-based assay affords a valuable technique for PML diagnosis and a versatile tool for the rapid scanning of NCCR mutations.