Nucleic acid-amplification testing (NAT) is not routinely practiced in blood banks from most low-income countries. We did an exploratory comparison of the performance of the standard immunoassay-based screening tests for the hepatitis B (HBV) and C (HCV) viruses with that of NAT, in blood donors. From January 1999 to March 2005, 94,806 blood donors were screened for anti-HCV antibodies and for hepatitis B surface antigen (HBsAg). Also, an exploratory period of molecular screening was carried out on 100 consecutive blood donors to detect HBV DNA and HCV RNA by home-made PCR techniques without sera pooling. In the 75-month period of serologic screening, HBsAg was detected in 219 donors (0.23%; 95% CI, 0.20- 0.26%) and anti-HCV antibodies in 922 (0.97%; 95% CI, 0.90-1.03%). The annual trend for HBsAg prevalence had a decreasing pattern over the years (p<0.001), whereas that for anti-HCV did not (p=0.19). In the molecular screening cohort, HBV DNA was detected in one donor (1%; 95% CI, 0-6%) and HCV RNA in another (1%; 95% CI, 0-6%). All these 100 donors tested negative to HBsAg and anti-HCV. Thus, the prevalence of positive results for HBV and HCV did not differ if considering immunoassays or NAT; nevertheless, these methods did not coincide in detecting HBV or HCV in the molecular screening cohort. In conclusion, NAT can detect cases of HBV and HCV infections that standard immunoassay techniques can not, even in a highly selected population at low risk, like blood donors. Large-scale studies are warranted for NAT to be considered as a systematic method for screening of HBV and HCV in Mexican blood banks.