AbstractTo compare the specificity and sensitivity of a real‐time fluorescent RT‐PCR assay with conventional RT‐PCR, sera from 110 healthy blood donors, 120 patients with a clinical diagnosis of chronic hepatitis B, and 416 patients with non‐A‐C acute hepatitis, as well as serial dilutions of HEV genotypes 1 and 4, were tested with both assays. All samples from healthy blood donors and patients with chronic hepatitis B were negative by both assays. Real‐time RT‐PCR could detect the same final dilution of genotype 1 as conventional RT‐PCR but could detect a 10‐fold lower concentration of genotype 4 than conventional RT‐PCR. Of 416 samples from patients with a clinical diagnosis of non‐A‐C acute hepatitis, 127 (30.5%) and 83 (20.0%) were positive for HEV by real‐time and conventional RT‐PCR, respectively. The concordance of real‐time and conventional RT‐PCR was 80.8%. Furthermore, 96 and 57 of 171 samples were positive for anti‐HEV IgM by real‐time and conventional RT‐PCR, respectively, and 31 and 26 of 245 samples negative for anti‐HEV IgM, were positive by real‐time and conventional RT‐PCR, respectively. All amplicons positive by conventional RT‐PCR were sequenced. Of 83 isolates, 7 and 76 belonged to genotypes 1 and 4, respectively. Thus, both assays have a high specificity, but the real‐time RT‐PCR assay is more sensitive than conventional RT‐PCR. Furthermore, HEV genotype 4 is responsible for most sporadic cases of hepatitis E in the north of China. J. Med. Virol. 79:1966–1973, 2007. © 2007 Wiley‐Liss, Inc.