Progressive multifocal leukoencephalopathy (PML) is a fatal disease caused by reactivation of JC polyomavirus (JCPyV) in immunosuppressed individuals and lytic infection by neurotropic JCPyV in glial cells. The exact content of neurotropic mutations within individual JCPyV strains has not been studied. We exploited the capacity of Single Molecule Real-Time sequencing technology to determine the sequence of complete JCPyV genomes in single reads. The method was used to precisely characterize individual neurotropic JCPyV strains of three PML patients without the bias caused by assembly of short sequence reads. In the cerebrospinal fluid sample (CSF) of a 73-year-old female (case 1) with rapid PML onset three distinct JCPyV populations could be identified. All viral populations were characterized by rearrangements within the noncoding regulatory region (NCCR) and one point mutation S267L in the VP1 gene, suggestive of neurotropic strains. One PML patient (case 2) had a single strain with small NCCR alterations, and one patient (case 3) had a single neurotropic strain with rearranged NCCR. This is the first report on full characterization of individual neurotropic JCPyV strains in the CSF of PML patients. It remains to be established whether PML pathogenesis is driven by one or several neurotropic strains in an individual.