The comb of the male is an important secondary sexual characteristic. Although quantitative trait loci (QTLs) related to comb size have been identified, molecular mechanisms underlying this trait remain mostly unknown. In this study, RNA sequencing (RNA-seq) was employed to compare whole transcriptomic differences between two groups of Partridge Shank chickens that are divergent in comb sizes. A total of 563 differentially expressed genes (DEGs) were identified, including 277 up-regulated and 286 down-regulated DEGs. According to the animal QTL database, eight DEGs including BMP2 and CHADL matching the reported QTLs were associated with the comb size. Functional annotation analysis revealed that DEGs were involved in cell communication and calcium signaling. Protein-protein interaction network analysis showed that STK32A, PIK3R1, EDN1, HSPA5, and HSPA8 have an impact on comb growth. Moreover, potential alternative splicing events and single nucleotide polymorphisms were also identified. Our data provide a source for identifying genes and pathways with functions critical to comb size and accelerate studies involving molecular mechanisms of this sexual ornament.