Objective To test the current progressive multifocal leukoencephalopathy (PML) diagnostic criteria by applying them to patients previously diagnosed with natalizumab (NTZ)-associated PML in a real-world clinical setting. Methods Patients from the Dutch-Belgian NTZ-PML cohort (n=28) were reviewed at the time of first diagnostic work-up and during follow-up, using the PML diagnostic criteria as proposed in a consensus statement from the American Academy of Neurology. Results At first diagnostic work-up, 18 patients (64.3%) met the criteria for high diagnostic certainty for PML (‘definite PML’ or ‘probable PML’). During follow-up, this increased to 20 patients (71.4%) as JC virus DNA was detected in cerebrospinal fluid of two additional patients. Nonetheless, 28.6% of patients were still classified as ‘possible PML’ or ‘not PML’ (6 (21.5%) and 2 (7.1%) patients, respectively) despite a very high suspicion for PML based on lesion evolution and signs of PML-immune reconstitution inflammatory syndrome on MRI, and development of compatible symptoms. Conclusions The current case definition of PML has low sensitivity for diagnosis of NTZ-PML in a real-world clinical setting in which MRI is frequently used for PML screening. This may delay diagnosis and appropriate management of PML, and may complicate a valid estimation of PML incidence during NTZ therapy.