Diseases that affect the upper respiratory tract in chelonians have been well described as a significant contributor to morbidity and mortality. Specifically, chelonian herpesvirus has been attributed to disease events in captive chelonians worldwide, but its importance on free-ranging populations is less well-known. Methods for the diagnosis of herpesvirus infections include histopathology, virus isolation, and conventional PCR. Real-time PCR has become an essential tool for nucleic acid quantitation. A quantitative real time TaqMan PCR assay was developed targeting the DNA polymerase gene of Terrapene herpesvirus 1 (TeHV1). This assay can detect as few as 10 viral copies per sample, which is over 100 times more sensitive than conventional PCR assays. Oral swabs from 396 free-ranging turtles from Tennessee and Illinois were collected and assayed for TeHV1. The overall prevalence of TeHV1 was 30.5% (n=121). Prevalence was highest in July (53.4%) compared to fall (33.8%) and spring (12.8%) (p<0.0001). The prevalence was found not to be variable across state (p=0.222), sex (p=0.380), or age class (p=0.106). The majority of positive turtles were found in edge habitats (55.0%), compared to forest (43.7%) and field (33.3%), which was not significantly different. This assay and its application can be utilized in free-ranging and captive box turtles for disease surveillance and progression of herpesviruses, and will aid in the characterization of the epidemiology of this disease.