To prevent unintentional transmission of bloodborne pathogens through organ transplantation, organ procurement organizations (OPOs) screen potential donors by serologic testing to identify human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infection. Newly acquired infection, however, may be undetectable by serologic testing. Our objective was to estimate the incidence of undetected infection among potential organ donors and to assess the significance of risk reductions conferred by nucleic acid testing (NAT) versus serology alone. We calculated prevalence of HIV and HCV—stratified by OPO risk designation—in 13 667 potential organ donors managed by 17 OPOs from 1/1/2004 to 7/1/2008. We calculated incidence of undetected infection using the incidence‐window period approach. The prevalence of HIV was 0.10% for normal risk potential donors and 0.50% for high risk potential donors; HCV prevalence was 3.45% and 18.20%, respectively. For HIV, the estimated incidence of undetected infection by serologic screening was 1 in 50 000 for normal risk potential donors and 1 in 11 000 for high risk potential donors; for HCV, undetected incidence by serologic screening was 1 in 5000 and 1 in 1000, respectively. Projected estimates of undetected infection with NAT screening versus serology alone suggest that NAT screening could significantly reduce the rate of undetected HCV for all donor risk strata. Results from this study suggest that the risk of undetected HCV infection among potential organ donors can be significantly reduced by screening with Nucleic Acid Testing as opposed to serology testing for both normal‐ and high‐risk donors. See editorial by Pruett on page 1115.