The purpose of this study was to identify and compare the presence of HCMV and EBV-1 in subgingival plaque, unstimulated saliva and peripheral blood of patients with chronic periodontitis. Forty patients diagnosed with chronic periodontitis (mean age, 41.7 years) were recruited. Unstimulated saliva, subgingival plaque and peripheral blood were collected from each patient and the DNA of each sample was isolated. The viruses were detected using the nested PCR technique. The detection frequency of EBV-1 in subgingival plaque, saliva and peripheral blood was 45%, 37.5% and 25%, respectively. HCMV was detected in 82.5% of subgingival plaque samples and peripheral blood and in 75% of salivary samples. The sensitivity for detecting EBV-1 in saliva and peripheral blood when EBV-1 was detected in subgingival plaque samples was low (22% and 27.7%, respectively) and the sensitivity for detecting HCMV in saliva and peripheral blood when compared to subgingival plaque was high (81.8% and 87.8%, respectively). There is a high agreement among the three sampling methods in detection of HCMV, but the detection of EBV-1 would require a combination of saliva and subgingival plaque sampling to avoid false negative results.