JC virus (JCV), a ubiquitous human polyomavirus, can cause fatal progressive multifocal leukoencephalopathy (PML) in immune compromised patients. The viral genome is composed of two conserved coding regions separated by a highly variable non-coding control region (NCCR). We analyzed the NCCR sequence from 10 PML JCV strains and found new mutations. Remarkably, the NCCR f section was mutated in most cases. We therefore explored the importance of this section in JCV expression in renal (HEK293H) and glioblastoma (U-87MG) cell lines, by adapting the emerging technology of DNA minicircles. Using bidirectional fluorescent reporters, we revealed that impaired NCCR-driven late expression in glioblastoma cells was restored by a short deletion overlapping e and f sections. This study evidenced a relevant link between JCV NCCR polymorphism and cell-type dependent expression. The use of DNA minicircles opens new insights for monitoring the impact of NCCR variation.