The aim of this study was to identify genes involved in comb development to provide insights into the molecular mechanism of chickens' comb formation. Fixation index (FST) and average number of base differences (π) of males with large and small combs were calculated based on whole-genome resequencing data. Chromosome regions with larger FST values and smaller π were considered candidate selection regions. Through further annotation of gene functions and pathways, we sought to screen possible selected genes associated with comb development. By screening whole genome resequencing data, FST and π were calculated using a 40 Kb sliding window strategy and eight regions were identified. Quantitative trait loci (QTL; FOX1 gene) related to comb length were found on chromosome 1. QTL (GLP1R, BTBD9, MIR6633, and MDGA1 genes) related to comb weight were found on chromosome 3. QTL (ALDH1A1, TMC1, and ANXA1 genes) associated with comb area were found on the Z chromosome. Nineteen genes, Wnt signaling pathway and neuroactive ligand-receptor interaction signaling pathway directly or indirectly related to comb growth and development were found through functional annotation and GO analysis. Among the selected genes LYN, GLP1R, FOX1, TBK1, STRAP, ST6GALNAC, and Wnt signaling pathways were related to immunity. MDGA1, BTBD9, MTSS1, SrGAPs, and neuroactive ligand receptor interaction signaling pathways related to neural function were screened. ALDH1A1, ANXAl, THBS, HIF-1α, and ACTN1 genes were related to heat dissipation. Among the selected genes FOX1, MDGAl, and ANXAl associated with immunity, neurological function, and heat dissipation function coincided with genes affecting the length, weight, and area of the comb. Comprehensive analysis suggested that comb development was due to multiple genes and signaling pathways.