The recent development of molecular diagnostic assays like as polymerase chain reaction (PCR) has provided powerful tools for the diagnosis of viral infection in the clinical fields. To ensure optimal therapeutic and prognostic value, it is important to establish whether viral load measurements are affected by repeated freeze-thaw (FT) cycles since the freezing of clinical samples is a universal method of specimen storage. This study was done to determine the effect of freezing and thawing of various samples on the quantitation and positivity of viral DNA. For this study, three different types of samples being used frequently in clinical fields were selected. Those samples contained ovine herpesvirus-2 (OvHV-2), a member of the gamma herpesviruses (genus Rhadinovirus). Two OvHV-2 DNA positive plasma samples, two peripheral blood mononuclear cell (PBMC) samples, and two nasal swab samples were randomly selected. They were carefully aliquated into 8 tubes for each sample. The aliquoted samples were frozen and thawed 0, 3, 6, 9, 12, 15, 18, and 21 times for each aliquot and then analyzed for changes on DNA levels and positivity. OvHV-2 DNA positivity and quantitation were tested by using nested PCR and real-time PCR, respectively. Twenty-one cycles of freezing and thawing did not significantly change this herpesviral DNA positivity in any of the samples tested. However, the decreases of viral DNA copies were observed in all samples by the increasing of FT cycles. In conclusion, the integrity of herpesviral DNAs in clinical specimens may be degraded by the increasing FT cycles. These results implicate that there is a need to aliquot specimen when it is first collected in order to reduce FT cycles during its analysis.