Herpes simplex virus (HSV) infections are efficiently treated with antiviral drugs such as acyclovir (ACV). However, resistance has been reported, mainly among immunocompromised patients (prevalence around 5%) and particularly allogeneic bone marrow transplant patients (prevalence reaching 30%). Resistance to ACV is associated with mutations on one of the two viral enzymes involved in the ACV mechanism of action: thymidine kinase (TK) and DNA polymerase. In 95% of the cases, ACV resistance is associated with a mutation in the TK gene as this enzyme is not essential for viral replication, unlike viral DNA polymerase, which is rarely involved in resistance. Strains resistant to ACV are almost always cross-resistant to other TK-dependent drugs such as penciclovir and famciclovir. Resistant infections can be managed by foscarnet or cidofovir but both are more toxic than ACV. These drugs also inhibit viral DNA polymerase but they are active on most ACV-resistant HSV as they do not depend on TK; nevertheless virus resistant to ACV because of a mutation in the DNA polymerase may be cross-resistant to these molecules. Published data on genetic characterization of resistant clinical isolates point out hot spots in viral TK and DNA polymerase genes. TK mutations associated with resistance are either insertion or deletion (codons 92 and 146 of TK gene) or substitution (codon 176-177, 336 of TK gene). DNA polymerase mutations are mainly located in conserved sites of the enzyme. A high level of gene polymorphism has also been reported for these genes, especially for TK. These results are useful for the development of rapid genotypic assays for the detection of mutations associated with resistance.