The herpes simplex virus (HSV) DNA polymerase is composed of the UL30 catalytic subunit and the UL42 processivity factor. The UL42 subunit increases the processivity of the polymerase along the DNA template during replication. The molecular mechanisms of HSV resistance to drugs interfering with viral DNA synthesis reported so far mainly rely on modifications of the viral thymidine kinase and DNA polymerase. We aimed to extensively describe the genetic variations of HSV UL42 processivity factor and to evaluate its potential involvement in resistance to antivirals. The full-length UL42 gene sequence of HSV was investigated among two laboratory strains (KOS and gHSV-2), 94 drug-sensitive clinical isolates and 25 phenotypically ACV-resistant clinical isolates. This work provided extensive data about natural variability of UL42 processivity factor among both HSV-1 and HSV-2 strains and showed that this viral protein is highly conserved among HSV strains, with a weaker variability for HSV-2. The analysis of 25 HSV clinical isolates exhibiting ACV-resistance documented most of the previously reported mutations related to UL42 natural polymorphism in addition to some unpreviously described polymorphisms. Surprisingly, a single-base deletion in UL42 gene sequence leading to a frameshift in the C-terminal region was identified among 3 HSV clinical isolates. From this preliminary study, UL42 processivity factor did not seem to be likely involved in HSV resistance to antivirals.