Although herpesviruses are known to contaminate the semen of several mammalian species, the occurrence of feline herpesvirus type 1 (FHV-1) in semen of infected cats has not been reported. Our objectives in this study were to investigate the presence of FHV-1 DNA in seminal fluid and frozen-thawed spermatozoa from FHV-1 infected Pallas' cats (Otocolobus manul) and assess the functionality of their frozen-thawed spermatozoa in vitro. Over a 3-yr period, semen (n = 33 ejaculates) was collected periodically via electroejaculation from four Pallas' cats chronically infected with FHV-1. Spermic ejaculates were frozen by pelleting on dry ice and stored in liquid nitrogen. After thawing, sperm motility and acrosome status were assessed over time during in vitro culture. For vitro fertilization (IVF), viable domestic cat (Felis silvestris catus) oocytes were inseminated with frozen-thawed Pallas' cat spermatozoa and evaluated for embryo cleavage. For FHV-1 polymerase chain reaction (PCR) analysis, DNA was extracted from seminal fluid, frozen-thawed spermatozoa, inseminated oocytes, heterologous IVF embryos, and conjunctival biopsies and analyzed for presence of a 322–base pair region of the FHV-1 thymidine kinase gene. Immediately post-thaw, sperm motility and percentage of intact acrosomes were decreased (P < 0.05) compared to fresh samples, and declined further (P < 0.05) during culture. However, all frozen-thawed IVF samples were capable of fertilizing domestic cat oocytes (overall, 46.1 ± 6.0% cleavage). PCR analysis did not identify FHV-1 DNA in any reproductive sample despite the repeated detection of FHV-1 DNA in conjunctival biopsies. These results suggest that semen collected from Pallas' cats infected with FHV-1 does not contain cell-associated or non–cell-associated virus and that frozen-thawed spermatozoa exhibit adequate function for potential genetic rescue with minimal risk of FHV-1 transmission.