Highly pathogenic avian influenza (HPAI) is a devastating viral disease that leads to severe pulmonary damage and high mortality rates in domestic poultry worldwide. It is caused by avian influenza type A viruses from the Orthomyxoviridae family. The HPAI virus contains a genome of eight negative-sense RNA segments. Among these, the PB2 (Polymerase basic protein) segment plays a crucial role in viral replication as a subunit of the viral RNA-dependent RNA polymerase (RdRp) complex. Targeting PB2 expression represents a potential strategy to control HPAI. MicroRNAs (miRNAs), which are short noncoding RNAs, play a significant role in post-transcriptional regulation of gene expression, influencing various biological processes including cell growth, tissue differentiation, apoptosis, and viral infection. Our previous research identified 200 differentially expressed pulmonary miRNAs in chickens during HPAI infection. In the current study, we screened these dysregulated miRNAs against the PB2 segment of the HPAI H5N1 virus. We evaluated parameters such as the thermodynamic stability of miRNA-mRNA complexes, complementarity of miRNA seed sequences with mRNA, conservation of the PB2 sequence, and accessibility of target sites to identify miRNAs that could potentially target the PB2 transcript of H5N1. Our findings indicate that chicken miRNAs gga-miR-17-3p, gga-miR-29a-5p, gga-miR-1718, gga-miR-16c-5p, and gga-miR-1744-5p are predicted to target the PB2 segment. This suggests that chickens possess inherent genetic potential to combat H5N1 infection, which can be leveraged to develop effective control strategies for HPAI in poultry and miRNA therapeutic potential.