Sequential herpes simplex virus type 1 (HSV-1) isolates were obtained from a paediatric haematopoietic stem cell transplant (HSCT) patient who received prolonged therapy with acyclovir (ACV) followed by foscarnet (PFA) and topical cidofovir (HPMPC) for severe persistent mucocutaneous HSV-1 infection. The isolates were retrospectively studied for drug resistance. The first resistant isolate associated with clinical failure of antiviral therapy emerged 44 days post-ACV treatment initiation. Susceptibility testing revealed an ACV-resistant HSV strain that demonstrated cross resistance to PFA in the absence of any previous PFA treatment. The observed cross resistance was conferred by a single amino acid substitution, Ser724Asn, in the HSV DNA polymerase (DNA pol) gene. During the subsequent course of ACV therapy, the ACV/PFA-cross-resistant isolates were replaced by ACV-resistant, PFA-sensitive isolates. These isolates carried no DNA pol mutations, but had an Arg163His substitution in the thymidine kinase gene. Upon subsequent switching of antiviral therapy from ACV to PFA, the original ACV/PFA-cross-resistant DNA pol mutant re-appeared. Our study shows the emergence of different drug-resistant HSV variants during ongoing, unchanged ACV therapy. Furthermore, a rapid re-selection of the original resistant variant was observed after switch. For optimal antiviral management of HSV infections in HSCT recipients, therapeutic decisions should be guided by drug susceptibility results whenever therapeutic failure is observed and/or when changes in antiviral treatment are considered.