Volunteers of Blood or who called donors are screened for hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV) infection by ELISA in the National center of blood bank in Baghdad. The risk of enzyme immunoassay (EIA) negative samples is not estimated till now in Iraq. The aim of this study is to screen the seronegative plasma of blood donors by a commercially available multiplex NAT test with mini-pooling. One thousands (1000) blood donors plasmas were screened by EIA and then NAT was performed on pools of ten blood plasma. A total of 100 minipools donors plasmas were screened by NAT. Positive HBV NAT minipools of plasma were (3%), 3 HCV (3%), and 3 (3%) HIV minipools by NAT. Accordingly, the use of NAT was shown to detect occult HBV and reduce window period in HCV and HIV in sero-negative plasma from blood donors. Therefore, the implementation of NAT with mini-poling for routine blood donor screening will provide a safe and cost-effective mean of screening for blood transfusion in Iraq.