Background: Sustained virologic response (SVR) to pegylated-interferon (PegIFN) and ribavirin (RBV) in hepatitis C virus (HCV)-infected patients could be predicted by detection of serum HCV RNA whereas detection of HCV RNA in other reservoirs such as peripheral blood mononuclear cells (PBMCs) for prediction of treatment response is still a mystery. Objectives: This study aimed at assessing the prediction of SVR by detection of HCV RNA in PBMCs or serum in patients during treatment. Methods: In a cohort study (2011 to 2014), 100 chronic HCV patients at Tehran Hepatitis Center were treated with PegIFN and RBV. Serum HCV RNA level was measured at baseline, 4, 12, and 24 weeks during treatment and at 24 weeks after termination of treatment. Meanwhile, HCV RNA was evaluated in PBMCs at weeks 4, 12, and 24 during the treatment. Results: Out of 100 patients treated in this study, 91 completed the course of treatment. Most patients were young males infected with HCV genotype 1. Cirrhosis and previous history of treatment was found in 16.5% and 26.5% of patients. Sustained virologic response was achieved in 65 (71.4%) patients. Among baseline parameters, only female gender was significantly associated with SVR. Undetectable serum HCV RNA at week 4 (OR = 4.74) and week 12 (OR = 11.63) of treatment predicted SVR rate while the same was not true for detection of serum HCV RNA at week 24 of treatment. Moreover, detection of HCV RNA in PBMCs at weeks 4 and 12 of treatment was not associated with the rate of SVR, while absence of HCV RNA in PBMCs at week 24 of treatment was associated with SVR (OR = 4.55). Conclusions: Detection of HCV RNA in PBMCs, especially at week 24 of treatment with PegIFN and RBV, could be considered as an additional marker for prediction of treatment response. It is recommended to assess HCV on-treatment kinetic in PBMCs of patients treated with direct-acting antiviral agents for prediction of treatment response.