Bovine herpes virus-1 (BoHV-1) is a serious viral pathogen of domestic and wild cattle. Herein, we report development of a new molecular diagnostic assay for rapid and sensitive detection of BoHV-1 utilizing the loop-mediated isothermal amplification (LAMP) technique. BoHV-1-LAMP assay was optimized to amplify the target DNA by incubation the Bst-DNA polymerase enzyme with a set of specially constructed six primers, based on the gE-gene of BoHV-1 virus, at 65 ◦C for 60 min. BoHV-1-LAMP products were detected by visual inspection using SYBR Green-I stain and had a ladder-like appearance by gel electrophoresis analysis. Negative results obtained with DNA from other tested fish viruses confirmed the specificity of the assay. The analytical sensitivity of the BoHV-1-LAMP assay was 1 fg of BoHV-1 DNA (dilution of 106). The developed assay could successfully detect BoVH-1 DNA from clinical samples. Results of this study indicate that the developed BoHV-1-LAMP is rapid and highly sensitive assay not only for detection of BoHV-1 in clinical samples, but also for differentiation between wild-type (gE-positive) and gE-negative BoHV-1 viruses, which will improve the control programs of BoHV-1 in Egypt. © 2014 Elsevier B.V. All rights reserved.