An outbreak of equine influenza was detected in Khyber Pakhtunkhwa during the years 2015-16. The isolated viruses were typed for the first time in Pakistan as H3N8 and grouped with the Clade 1 viruses of Florida sub-lineages on the basis of Hemagglutinin (HA) and Neuraminidase (NA) gene sequence analysis. Here we described the genetic analysis of two internal genes (Nucleoprotein and Matrix) of the Pakistani isolates. The primer pairs were designed to amplify the NP and M internal genes of the 19 equine H3N8 influenza A viruses identified and isolated in this outbreak. Phylogenetic analysis of the derived sequences was performed and compared with the contemporary available sequences online. Comparison of the nucleotide sequences of the two internal genes of the Pakistani strains showed high similarity (99.7-100%) with the avian influenza A viruses (Avian/Pakistan/H7N3/ 2004) isolated in Pakistan. Two major substitutions i.e. F63L and K243R were recorded in Matrix gene of the isolates from the closely related Chicken/Pakistan/04 H7N3 virus. Notably NP gene did not acquire any amino acid substitution on comparison with Chicken/Pakistan/04. All isolates shared 99.5-99.9% homology with the reference sequences on alignment for M1 and NP amino acid sequences, respectively. Phylogenetic analysis revealed clustering of the Pakistani isolates with the avian viruses isolated in Pakistan in previous outbreaks in commercial and backyard poultry. Epidemiological investigation of the outbreak suggested that reassortment of equine H3N8 viruses with avian Influenza A H7N3 most probably, might be due to the mix farming system here. In conclusion, Phylogenetic analysis of the internal genes reported here will be helpful in early detection and control of EI.