(1) Background: Feline herpesvirus (FHV-1) is a significant pathogen in cats, causing respiratory and ocular diseases with consequential economic and welfare implications. (2) Methods: This study aimed to isolate and characterize FHV-1 from clinical samples and assess its pathogenicity. We collected 35 nasal and ocular swabs from cats showing symptoms of upper respiratory tract infection and FHV positivity detected by polymerase chain reaction (PCR). Viral isolation was carried out using feline kidney (F81) cell lines. Confirmation of FHV-1 presence was achieved through PCR detection, sequencing, electron microscopy, and indirect immunofluorescence assay. The isolated strains were further characterized by evaluating their titers, growth kinetics, and genetic characteristics. Additionally, we assessed the pathogenicity of the isolated strains in a feline model, monitoring clinical signs, viral shedding, and histopathological changes. (3) Results: Three strains of FHV-1 were isolated, purified, and identified. The isolated FHV-1 strains exhibited high homology among themselves and with domestic isolates and FHV-1 viruses from around the world. However, they showed varying degrees of virulence, with one strain (FHV-A1) causing severe clinical signs and histopathological lesions. (4) Conclusion: This study advances our understanding of the genetic and pathogenic characteristics of FHV-1 in China. These findings underscore FHV-A1 isolate as a potentially ideal candidate for establishing a challenge model and as a potential vaccine strain for vaccine development.