The duration-of-fertility (DF), which was defined as the number of days when breeding hens lay fertile eggs following copulation or artificial insemination (AI), is an important economic trait in chick production when it has strong effects on fertile egg output and production costs. Little is known about the underlying genes and molecular markers related to DF trait to date. Here, we measured the DF of 701 Chinese Jinghong hens and 408 Jingfen hens. The DF showed high individual variability and potential for genetic improvement. Then, 192 Jinghong breeding hens were provided for a genome-wide association study, 27 SNPs respectively located in three genomic linkage regions (GGA1:41Kb; GGA3:39Kb and GGA8:39Kb) were suggested to be significantly associated with DF. Particularly, 6 of these 27 SNPs were further verified to be associated with DF in the 701 Jinghong and 408 Jingfen hens using PCR-RFLP genotyping method. These 27 SNPs were also mapped to 7 genes according to their genomic position. Furtherly, 5 of these 7 genes were tested using qPCR. Results show that the CYP2D6, WBP2NL, ESR1 and TGFBR3 mRNA expression levels of hens with long DF were significantly higher than the hens with short DF (P < 0.05). Overall, findings in our research provide new insight into the genetic basis of duration-of-fertility in breeding hens while providing new clues for further functional validation on the DF-related genetic regulation mechanism and improvement of DF through chicken breeding.