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The eggshell quality traits in avian species, including Yunnong chickens (YN), are crucial for commercial and breeding purposes. The use of advanced biotechnologies, such as gene sequencing, has become increasingly common for identifying genetic markers and comparing specific traits in livestock. However, genetic markers related to eggshell traits in YN chickens remain unexplored. This study aimed to compare eggshell quality traits and identify genetic markers in YN chickens versus commercial Jingfen chickens (JF). A total of 400 chicks (200 per breed) were reared for 300 days under controlled conditions. At the end, 60 eggs from each breed were analyzed using scanning electron microscopy to examine eggshell cross-sections and mammillary layer structure. Tissue samples from kidneys and eggshell glands were collected from six hens per breed for RT-qPCR analysis to study gene expression profiles. Results showed that YN chickens had significantly higher eggshell strength, thickness, mammillary density, and effective layer thickness than JF chickens (p < 0.01). Despite this, YN chickens had a thinner mammillary layer. RT-qPCR analysis confirmed five candidate genes related to eggshells (LSS, NSDHL, MSMO1, SQLE, and FDFT1) that play an important role in this process. Pathway analysis revealed significant enrichment in several biological processes, including steroid biosynthesis, glycerolipid metabolism, purine metabolism, and thiamine metabolism. Based on these results, the YN chickens have strong eggshells with better thickness, mammillary density, and effective layer thickness. In addition, genomic and pathways analysis suggest a complex interaction of metabolic, immune, and cellular processes potentially influencing eggshell quality traits in poultry. Full article