The highly pathogenic avian influenza (HPAI)-H5N1 virus is a highly contagious evolving pathogen that spreads rapidly among poultry sectors causes a threat to the poultry industry globally. H5N1 viruses of clade 2.3.4.4b have been widely circulating in wild-domestic birds and detected in Europe, Africa, North America, and Asia since October 2020. In this study, 40 tissue and swabs specimens were collected from 25 poultry flocks of different localities in New Valley Governorate, Egypt during 2023-2025. The collected samples were tested for H5N1, H9N2, H5N8, and H6N2 AIV subtypes as well as other pathogens such as NDV, IBV, ILT, and IBDV. To the best of our knowledge, this is first detection of HPAI-H5N1 of clade 2.3.4.4b viruses in broilers in New Valley Governorate, Egypt. Five positive H5N1 specimens were isolated in specific-pathogen-free-embryonated chicken eggs (SPF-ECE) with positive HA titers of 7–8 HA units. Using real-time reverse transcriptase polymerase chain reaction (RT-qPCR) assay targeting the M gene, only 4 samples were considered positive. Collectively, four samples were identified as positive for AIV of the H5N1 subtype through a one-step PCR assay, whereas the other samples were negative for other tested pathogens. Partial sequencing and the phylogenetic analysis of HA gene segment confirmed that the NewValley-1-H5N1-2023, and NewValley 2-H5N1-2024 isolates belonged to the H5N1 subtype of clade 2.3.4.4b. Our HPAI-H5N1 strains shared genetic similarity with the HPAI-H5N1 strains that reported in Europe, Asia, and Africa during 2021–2022. Currently, our H5N1 strains shows a genetic similarity to HPAIV-H5N1of clade 2.3.4.4b Egyptian isolates with nucleotide and amino acid identities percentage (96%-99%) meanwhile, they shared a low genomic relatedness (72%-84%) with commonly available vaccine. Genetically, the current H5N1 strains had R72S in the receptor binding sites of the HA protein of Egyptian H5N1 and had amino acid mutations in the HA immunogenic epitopes (A83D, T140A). Continuous monitoring and early determination of HPAI-H5N1 viruses in New Valley Governorate, Egypt as well as potentially updating the vaccine seed in a timely manner is essential to ensure the effectiveness of the vaccine as a control strategy of HPAI-H5 virus in the poultry industry.