Emergence of acyclovir (Acy)-resistant herpes simplex virus (HSV) is a major concern in bone marrow transplant recipients. Phenotypic and genetic characterization of thymidine kinase (TK) was done for 7 Acy-susceptible and 11 Acy-resistant HSV-1 isolated from 11 patients. In total, 19 amino acid substitutions were detected that were not related to Acy resistance but to TK gene polymorphism, including 5 mutations that have not been previously reported. The Acy-resistant strain from 1 patient presented no TK gene mutation related to resistance. Five patients (45%) had isolates that harbored point mutations leading to amino acid substitutions that could be associated with Acy resistance. Of the 5 substitutions detected, 3 have not been previously reported (codons 51, 83, and 175). A nucleotide insertion or deletion was detected in resistant isolates from 5 patients (45%); these mutations are located in homopolymer repeats at codon 92 (1 subject) and at codon 146 (4 subjects).