Diffuse malignant mesothelioma (DMM) is increasing in incidence on a worldwide basis and is linked to exposure to asbestos. Simian virus 40 (SV40), a DNA virus, was introduced inadvertently to human populations through contaminated polio vaccine during the years 1956-1963. It has been associated with various types of malignancy in animal experiments. There have been suggestions that SV40 might play a role in the pathogenesis of DMM. To evaluate the association between SV40 and DMM in Iranian patients. In a case-control study between the years 2007-2008, isolated DNA from 60 paraffin blocks of patients with DMM and 60 controls was assessed to detect three human polyomaviruses (JCV, BKV, and SV40) using three different sets of primers by multiplex nested PCR analysis. We related the patients with diffuse malignant mesothelioma to possible sites of exposure to asbestos. None of the DMMs nor any patient in the control group had SV40 genome on polymerase chain reaction (PCR). All of the cases were SV40 T antigen negative. This study suggests that DMM is independent of SV40 infection in Iran.