The diagnosis of natalizumab-associated progressive multifocal leukoencephalopathy (PML) in multiple sclerosis patients in an asymptomatic stage is crucial since it is associated with better clinical outcome measures. Current diagnostic criteria on PML diagnosis in asymptomatic patients require the detection of JC virus and corresponding imaging findings. Magnetic resonance imaging (MRI) is the most sensitive diagnostic method for these purposes. However, the diagnosis of asymptomatic natalizumab-associated PML based on MRI findings can be challenging particularly in case of inconclusive or negative results on JC virus detection in the cerebrospinal fluid. In this report, we present a case series demonstrating different diagnostic scenarios of asymptomatic PML diagnosis based on MRI findings in combination with inconclusive or negative results on JC virus detection in the cerebrospinal fluid. We discuss the challenges of applying current PML diagnostic criteria in asymptomatic natalizumab-associated PML patients and stress the need for specific diagnostic criteria and guidelines regarding managing these diagnostic dilemmas in order to facilitate an early and correct diagnosis of PML presumably leading to a better clinical outcome.