Newcastle disease (ND) is a fatal and contagious disease that poses a constant threat to the poultry industry around the globe. Due to the complex clinico-pathological picture and high genetic variability, the efficient diagnosis of NDV strains is a challenge. In an emerging wave of ND in the north of Pakistan, samples from six outbreaks in commercial poultry and two from healthy backyard poultry flocks were screened for NDV. A real-time PCR based on the fusion and polymerase genes of NDV detected all six isolates whereas a validated real-time PCR based on the matrix gene failed to detect any of these isolates, most likely due to substantial mismatches in the probe-binding site. All isolates have shown ICPI and MDT values similar to the velogenic form of NDV strains. The cleavage site in the F protein was found to be ¹¹²RRQKR↓F¹¹⁷, typical of virulent NDV. Phylogenetic reconstruction, based on fusion and matrix genes, provided enough evidences to consider these isolates as a new subgenotype within genotype VII. This study raised concerns about the genetic variability of NDV circulating in Pakistan, and sensitivity of the assays for the detection of the NDV isolates in clinical samples.