The emergence of hepatitis B virus (HBV) drug-resistant (and multidrug-resistant) strains during long-term therapy with nucleoside/nucleotide analogues is associated with treatment failure and, therefore, represents a clinical challenge. For clinicians, the close monitoring and management of resistance has become a key issue in clinical practice. For HBV virologists, the understanding of the mechanism of emergence of specific mutant strains in the viral quasispecies during treatment is also an important issue. If a particular viral strain can emerge in the quasispecies within a particular environment, it is probably because its fitness is superior to other strains. The present review focuses on viral fitness as well as viral infectivity, and in particular on technical means that are available to study this viral fitness in vitro and in animal models.