The aim of our study was to investigate B cell clonality of distinct localizations of primary central nervous system lymphomas (PCNSL) in AIDS patients. We selected seven autoptic cases of AIDS PCNSL with at least two separated localizations of the disease in the central nervous system (CNS). Clonality was investigated by immunoglobulin light chain restriction and by semi-nested PCR for the immunoglobulin heavy chain (IgH). Three cases showed a concordant restriction in the two lesions. In an additional case, an identical IgH sequence was found. The remaining three cases did not provide consistent information because of extensive necrosis in the lesions. Our findings suggest that multifocality of PCNSL is due to dissemination of a single neoplastic clone rather than to a synchronous transformation of multiple B cell clones.