Bovine herpesvirus 1 (BoHV-1) is an evident respiratory and reproductive pathogen in bovines. It causes infectious bovine rhinotracitis (IBR) and reproductive disorders. Present study evaluated the prevalence of BoHV-1 among the healthy and sick cattle and buffaloes. Two hundred blood samples were randomly collected from the animals of district Okara, Pakistan. To detect BoHV-1 antibodies, indirect ELISA was performed. Out of 200 samples from both species (cattle and buffalo), an overall prevalence of BoHV-1 of 124/200 (62%) was obtained. Samples from buffalo had higher species-specific prevalence 64/98 (65%) than samples from cattle 60/102 (58.8%). Overall, BoHV-1 was more prevalent in female (73.35%) animals as compared to male animals (48.5%). Present study depicted that 84/124 (67.70%) animals from positive results were apparently healthy and 40/124 (32.25%) animals from seropositive results were sick. Identification and molecular characterization was performed on sixty seropositive blood samples, PCR was performed by using glycoprotein E gene primers on isolated DNA from these samples, six were positive 6/60 (10%), and gene sequencing and phylogenetic analysis had shown close similarities with other BoHV-1 gE gene sequences world widely. The accession number assigned to these samples in GenBank were OQ656376.1 and OQ656377.1. Subtyping of BoHV-1 was conducted by multiplex PCR following HindIII enzyme activity, three samples were identified as BoHV-1.1 that indicated BoHV1.1 prevalence in study area. In this study, Multiplex PCR proven as efficient, economical and rapid technique for diagnosis and subtyping. This study will be helpful in vaccine development and in devising measures to control the spread of BoHV-1.