The polyomaviruses KI (KIPyV) and WU (WUPyV) were recently identified mainly in respiratory samples from children and immunosuppressed patients. Investigation of 54 autopsy brain tissue samples from 22 subjects demonstrated that WUPyV DNA and KIPyV DNA could be detected in 1 of 4 human immunodeficiency virus (HIV)-positive individuals with progressive multifocal leukoencephalopathy (PML) and in 3 of 10 HIV-positive individuals without PML, but not in 8 HIV-negative individuals. Viruses were localized in all regions of the central nervous system that were analyzed, that is, the cerebral hemispheres, cerebellum, pons, and medulla oblongata. No specific histopathological findings were found to be associated with the presence of WUPyV and KIPyV.