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Background Infections due to drug-resistant herpes simplex viruses (HSV) represent an important clinical concern, especially in immunocompromised patients. The present study was aimed at detecting acyclovir (ACV) susceptibility in HSV clinical samples.Methods A total of 13 HSV-positive clinical samples (5 HSV-1 and 8 HSV-2) recovered from patients (1 immunocompromised and 12 of unknown immune status) were included in the study. The genotypic analysis involved an initial UL23 (thymidine kinase) gene sequencing, followed by a confirmatory phenotypic assay using plaque reduction technique.Results Two novel amino acid changes, A37V and H283N, were detected in HSV-1 positive clinical samples, which were found to be susceptible to acyclovir (half maximal effective concentration = 1.5 µM) by plaque reduction assay.Conclusions These two novel amino acid changes could be therefore considered as natural polymorphisms, a phenomenon widely associated with the HSV-UL23 gene.