_1757587371 20250911104251000 Rovedar Daryoushbabazadeh@gmail.com Rovedar Journal of World’s Poultry Science J. World's Poult. Sci. 2980-7999 09 01 2025 4 3 Umar Aziz https://orcid.org/0009-0009-6266-4340 Abdul Rehman https://orcid.org/0009-0009-3221-2984 Muhammad Mushahid https://orcid.org/0000-0001-8670-3011 Fasih Ur Rehman https://orcid.org/0000-0002-7750-3453 Nauman Khan https://orcid.org/0009-0008-1289-6859 Muhammad Hanzalah Yousaf https://orcid.org/0009-0005-7241-5701 M Khuzema Niaz https://orcid.org/0009-0009-2039-3570 Muhammad Arslan Akbar https://orcid.org/0000-0001-5418-1271 Muhammad Rizwan https://orcid.org/0009-0004-9832-9193 Saleh Ahmad https://orcid.org/0009-0007-0595-4320 Introduction: Poultry layer breeds have undergone extensive selection for egg production traits, yet the genetic basis of many economically important characteristics remains incompletely understood. In the present study, a comprehensive comparative genomic analysis of multiple commercial and indigenous poultry layer breeds was conducted to identify novel genes associated with egg production and disease resistance, such as viral and bacterial infections, particularly through innate immune pathways.Materials and methods: A comprehensive comparative genomic analysis was conducted using whole-genome sequencing data from 135 individuals, including 30 commercial layers (White leghorn and Rhode Island red), 90 indigenous chickens from six local breeds, and 15 red jungle fowl representing the ancestral population. Using fixation index scans, haplotype-based selection analysis, and Tajima’s D, genomic regions under positive selection were identified. By integrating gene prediction tools and protein function analysis, 12 novel genes in layer breeds with strong selection signals and potential roles in egg production and immune response were identified.Results: In silico functional analysis, utilizing protein domain annotation, structural modeling, and pathway enrichment, suggested that these novel genes are involved in eggshell formation, egg production, immune response, and metabolic regulation. Notably, GALLUS-NOVEL-3, a previously uncharacterized gene with high predicted oviduct expression and strong selection signatures in commercial layers, may play a role in calcium transport and eggshell mineralization. Protein structure prediction and domain analysis further supported the potential functionality of these novel genes, revealing conserved features linked to reproductive physiology and immune defense.       Conclusion: The present findings provided new insight into the genetic basis of economically important traits in layer chickens and highlight promising targets for breeding programs aimed at improving egg production and disease resistance. 09 01 2025 30 42 https://creativecommons.org/licenses/by/4.0 10.58803/jwps.v4i3.79 https://jwps.rovedar.com/index.php/JWPS/article/view/79 https://jwps.rovedar.com/index.php/JWPS/article/download/79/93 https://jwps.rovedar.com/index.php/JWPS/article/download/79/93 10.1101/gr.4141805 10.1371/journal.pgen.1007989 10.1017/S0043933912000360 10.2527/af.2016-0004 10.1186/s12863-019-0771-7 10.1093/ps/85.2.227 10.1038/s41576-018-0082-2 10.1038/nrg1294 10.1038/nature08832 10.1016/j.crvi.2010.12.012 Minga UM, Msoffe PL, and Gwakisa PS. 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