Newcastle disease (ND) is a highly contagious disease of many avian species and is particularly responsible for devastating disease outbreaks in commercial poultry flocks in Pakistan that incur huge economic losses to the national poultry industry annually. Despite implementation of an extensive vaccination program for poultry birds, the disease appears in an endemic form in commercial broiler and layer poultry farms. This study was conducted to identify the prevalent velogenic NDV strain responsible for disease outbreaks in commercial poultry farms in Punjab, Pakistan. The NDV strains isolated from pathological specimens through inoculation in embryonated chicken eggs were characterized biologically through the intracerebral pathogenicity index (ICPI), and genetically on the basis of the fusion (F) protein cleavage site. Among these, six NDV isolates showed an F protein cleavage site motif (¹¹²RRQKRF¹¹⁷) and an ICPI value ranging between 1.5 and 1.88, both are characteristics for velogenic strains of NDV. In addition, phylogenetic analysis based on a partial sequence of the F protein gene clustered these isolates within class II, genotype VII and specifically within genotype VII-e. This is the first report that demonstrated the presence of such NDV strains in commercial poultry farms in northern Punjab of Pakistan.