The age of onset of sexual maturity is an important reproductive trait in chickens. In this study, we explored candidate genes associated with sexual maturity and ovary development in chickens. We performed DGE RNA-sequencing analyses of ovaries of pre-laying (P-F-O1, L-F-O1) and laying (P-F-O2, L-F-O2) hens of two sub-breeds of Ningdu Yellow chicken. A total of 3197 genes were identified in the two comparisons, and 966 and 1860 genes were detected exclusively in comparisons of P-F-O1 vs. P-F-O2 and L-F-O1 vs. L-F-O2, respectively. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that genes involved in transmembrane signaling receptor activity, cell adhesion, developmental processes, the neuroactive ligand–receptor interaction pathway, and the calcium signaling pathway were enriched in both comparisons. Genes on these pathways, including growth hormone (GH), integrin subunit beta 3 (ITGB3), thyroid stimulating hormone subunit beta (TSHB), prolactin (PRL), and transforming growth factor beta 3 (TGFB3), play indispensable roles in sexual maturity. As a gene unique to poultry, hen egg protein 21 kDa (HEP21) was chosen as the candidate gene. Differential expression and association analyses were performed. RNA-seq data and qPCR showed that HEP21 was significantly differentially expressed in pre-pubertal and pubertal ovaries. A total of 23 variations were detected in HEP21. Association analyses of single nucleotide polymorphisms (SNPs) in HEP21 and reproductive traits showed that rs315156783 was significantly related to comb height at 84 and 91 days. These results indicate that HEP21 is a candidate gene for sexual maturity in chickens. Our results contribute to a more comprehensive understanding of sexual maturity and reproduction in chickens.