AIM: To compare the sequencing of PCR products, pyrosequencing, and real-time PCR for detection of Tyrosinemethionine-aspartate-aspartate (YMDD) mutants in patients with chronic hepatitis B.METHODS: Mixtures of plasmids and serum samples from 69 chronic hepatitis B patients treated with lamivudine were tested for YMDD mutations by sequencing of PCR products, pyrosequencing, and real-time PCR, respectively. Time required and reagent costs of the three assays were evaluated.RESULTS: Real-time PCR detected 100%, 50%, 10%,1% and 0.1% of YVDD plasmid in mixtures with 106 copies/mL of YMDD plasmid, whereas sequencing and pyrosequencing only detected 100% and 50% of YVDD plasmid in aliquots of the corresponding mixtures. Completely concordant results were obtained from 60 (87%)out of the 69 clinical serum samples by the three assays.Mutants were detected by real-time PCR in less than 20% of the total virus population, but no mutant was detected by sequencing and pyrosequencing. In addition,real-time PCR required less time and was more cost-effective than the other two assays. However, throughput of pyrosequencing was the highest.CONCLUSION: Among the three assays compared,real-time PCR is the most sensitive, cost-effective, and time saving for monitoring YMDD mutants in patients with chronic hepatitis B on lamivudine therapy.