Primary structures of DNA polymerase (ul30) and thymidine kinase (ul23) genes from several herpes simplex virus type 1 (HSV-1) clinical isolates differing in sensitivity to several antiherpetic drugs were determined and compared to those of two laboratory HSV-1 strains one of which (L2) was sensitive and the other (L2/R) was resistant to acyclovir. The phylogenetic sequence analysis showed that the ul30 and ul23 sequences of clinical isolates were close to those of L2, and that ul30 conserved regions differed between HSV-1 isolates and L2only in point mutations and degenerated substitutions. Several new mutations in the HSV-1 DNA polymerase and thymidine kinase functional domains were identified as substitutions associated with strain resistance to ACV and other antiherpetic drugs. © 2010 Pleiades Publishing, Ltd.