The storage of biological samples may affect detection of viral nucleic acid, yet the stability of viral nucleic acid at standard laboratory storage temperatures (-70° C and -20° C) has not been comprehensively assessed. Deterioration of viral RNA and DNA during storage may affect the detection of viruses, thus leading to an increased likelihood of false-negative results on diagnostic testing. The viral loads of 99 hepatitis C virus (HCV), 41 HIV, and 101 hepatitis B virus (HBV) patient samples were measured before and after storage at -20° C and -70° C for up to 9.1 years using Versant branched DNA assays, Cobas Monitor assays, and/or AmpliPrep/AmpliScreen assays. Clinical samples stored at -20° C for up to 1.2 years and at -70° C for up to 9 years showed a statistically significant difference from baseline with respect to HCV RNA titer, although this difference was not greater than 0.5 log 10 unit. The concentration of HIV RNA in clinical samples stored at -20° C for 2.3 years and at -70° C for up to 9.1 years did not differ significantly from the baseline viral load. HBV DNA-positive clinical samples stored at -20° C for up to 5 years and at -70° C for up to 4 years differed significantly in viral load. In all studies, however, the loss of viral load of HCV, HIV, or HBV in clinical samples tested after storage at -20° C and -70° C for up to 9 years ranged from 0.01 to 0.35 log 10 IU/ml and did not exceed 0.5 log 10, which is the estimated intra-assay variation for molecular tests. Hence, the loss was considered of minimal clinical impact and adequate for the detection of HCV, HIV-1, and HBV nucleic acids using nucleic acid assays for the assessment of the infectious risk of cell, blood, and tissue donors.