The present study investigated the prevalence of infection by JC and BK polyomaviruses (JCV and BKV) in patients with chronic renal disease (CRD), kidney transplant recipients, and a control group of asymptomatic subjects. We tested a total of 295 urine samples. After DNA extraction, polymerase chain reaction assay was used to amplify a fragment of 173 bp of the polyomavirus T antigen, followed by analysis using the BamHI restriction endonuclease. Infection by polyomavirus was detected in 17.6% (52/295 subjects) of the subjects. Whereas 30.5% (18/59) of transplant recipients were infected, the frequency was only 22.4% (30/134) in the control subjects, and 3.9% (4/102) in the CRD group (all JCV). The vast majority of infections (88.9%; 16/18) in transplant recipients were of the BKV type, whereas this type was absent in CRD patients, and made up only 10.0% (3/30) of infections in the control group. The risk of BKV infection was 72 times greater in renal transplant patients than in asymptomatic subjects. The low frequency of infection found in CRD patients may have been related to elevated levels of urea excreted in the urine, together with reduced urine volume and cell content. These factors may combine to reduce viral load or inhibit amplification. The results of the study indicate a need for the routine screening for polyomavirus in pre- and post-transplant patients, as well as organ donors, considering that BKV infection has been associated with graft rejection in kidney transplants.