Viral differences among lamivudine resistant hepatitis B (HBV) genotypes have not been yet investigated. Therefore, we analyzed the characteristics of these viral strains in vivo. Forty-one patients carrying lamivudine resistant HBV were enrolled. Twenty-six patients (63%) carried resistant HBV genotype A (group A) and 15 patients (37%) carried resistant HBV genotype D (group D). The rate of reverse transcriptase 204I mutants was significantly higher in group D (67%) compared with group A (19%), whereas rt204V mutants (81% in group A vs 33% in group D; P = .006) and rt180M mutants (81% in group A vs 40% in group D, P = .015) prevailed in group A. The median time of shift from rt204I to rt204V mutants was significantly shorter in group A (4 months in group A, >12 months in group D, P < .001). Additional resistance associated mutations were detected exclusively in group D (P = .004). In a multivariate analysis, HBV genotype (P = .039) and pretreatment serum HBV DNA (P = .001) were independently associated with emerging rt204I or rt204V mutants, respectively. Serum HBV copy numbers after emergence of resistance were higher in group A (mean log10 6.99 copies/ml; range 3–9) compared with group D (mean log10 6.1 copies/ml; range 3.3–8; P = .04). There was no difference between both groups regarding core promoter/precore mutations, viral turnover, and number of flares or disease progression during follow-up. In conclusion, the mutational pattern during selection of lamivudine resistant HBV strains differs between genotypes A and D. This may have consequences for a salvage regimen initiated for treatment of lamivudine resistant HBV. (HEPATOLOGY 2004;39:42–50.)