The objective of this study was to develop a novel EvaGreen (EG) based real-time PCR technique for the simultaneous detection of Equine herpesvirus 1 (EHV-1) and Equine herpesvirus 4 (EHV-4) genomes from equine nasal swabs. Viral genomes were identified based on their specific melting temperatures (T (m)), which are 88.0 and 84.4 A degrees C for EHV-1 and EHV-4, respectively. The detection limitation of this method was 50 copies/mu l or 0.15 pg/mu l for EHV-1 and 5 copies/mu l or 2.5 fg/mu l for EHV-4. This assay was 50-1,000 times more sensitive than the SYBR Green (SG)-based assay using the same primer pairs and as sensitive as the TaqMan-MGB probe-based assay. The validity of the real-time PCR assays was confirmed by testing 13 clinical samples. When all results of the EG, SG, and TaqMan probe-based singleplex and duplex real-time PCRs were considered together, a total of 84.6 % (11/13) horses and donkeys were positive for at least one virus. EHV-1 and EHV-4 coexisted in 81.8 % (9/11) horses. Overall, we report that the EvaGreen duplex real-time PCR is an economical and alternative diagnostic method for the rapid differentiation of EHV-1 and EHV-4 in nasal swabs.