HBV infection is contagious and may be transmitted vertically or horizontally by blood products and bodysecretions. Over 50% of Iranian carriers have contracted the infection prenatally, making this the most likelyroute of transmission of HBV in Iran. This study assesses the resistance to Lamivudine in patients with chronichepatitis B infection using a new ZNA probe Real Time PCR method. To evaluate the effectiveness of Lamivudinetherapy for chronic hepatitis B infection, a study was conducted on 70 patients (63 men and 7women), who hadreceived the drug first line. All patients were tested for the presence of HBsAg and HBeAg, the serum ALT leveland the HBV DNA load before and after treatment. In all samples resistance to Lamivudine was tested with theZNA Probe. Our results showed that ZNA Probe Real Time PCR method could detect wild type,YMDD, and itsmutants, tyrosine-isoleucine-aspartate-aspartate and tyrosine-valine-aspartate-Aspartate. Among an estimatedseventy patients with chronic hepatitis B infection, 18 (25.7%) were resistant to lamivudine. Only one patientwas negative for presence of HBS-Ag (5.6%) and two patients were negative for HBe-Ag (11.1%). Real-timePCR with Zip nucleic acid probes is a sensitive, specific and rapid detection method for mutations in the YMDDmotif, which will be essential for monitoring patients undergoing Lamivudine antiviral therapy.