Herpesvirus infections have been associated with high morbidity and mortality in populations of captive emydid chelonians worldwide, but novel herpesviruses have also recently been identified in apparently healthy free-ranging emydid populations. Blanding's turtle (Emydoidea blandingii), an endangered species in Illinois, has experienced range-wide declines because of habitat loss, degradation, and fragmentation. A novel herpesvirus, Emydoidea herpesvirus 1 (EBHV1), was identified in Blanding's turtles in DuPage County, IL, in 2015. Combined oral-cloacal swabs were collected from radio transmitter–fitted and trapped (n = 54) turtles multiple times over the 2016 activity season. In addition, swabs were collected at a single time point from trapped and incidentally captured (n = 84) Blanding's turtles in DuPage (n = 33) and Lake (n = 51) counties over the same field season. Each sample was tested for EBHV1 using quantitative polymerase chain reaction (qPCR). EBHV1 was detected in 15 adult females for an overall prevalence of 10.8% (n = 15/138; 95% confidence interval [CI]: 6.2–17.3%). In radio transmitter–fitted females, there was a significantly higher prevalence of EBHV1 DNA in May (23.8%, n = 10/42) than June (3.6%, n = 1/28), July (0%, n = 0/42), August (0%, n = 0/47), or September (7.7%, n = 3/39) (odds ratio: 12.19; 95% CI: 3.60–41.30). The peak in May corresponds to the onset of nesting and may be associated with increased physiologic demands. Furthermore, all positive turtles were qPCR negative in subsequent months. There were no clinical signs associated with EBHV1 detection. This investigation is the critical first step to characterizing the implications of EBHV1 for Blanding's turtle population health and identifying management changes that may improve sustainability.