The function of the sperm storage tubules is directly correlated with the fertility of laying hens. However, little is known about the molecular mechanisms regulating the fertility traits in chicken. To identify genetic markers associated with reproductive traits, we calculated fertility rate at 61 to 69 wk (51 D) of Jing Hong chickens parent generation as the phenotype and the genotype were detected by the chicken 600K Affymetrix Axiom High Density single nucleotide polymorphisms (
SNP
)-array. The genome-wide association study using 190 Jing Hong hens showed that the 20 SNP in chromosomes 3 and 13 were significantly associated with fertility rate. To verify these results, a total of 1900 Jing Hong laying hens from 2 populations (P1 and P2) were further genotyped by polymerase chain reaction–restriction fragments length polymorphisms method. The association analysis results revealed that 12 polymorphisms (AX-75769978, AX-76582632, AX-75730546, AX-75730496, AX-75730588, AX-76530282, AX-76530329, AX-76529310, AX-75769906, AX-75755394, AX-80813697 and AX-76582809) out of 20 showed highly significant effects (
P
< 0.0001) on fertility rate in P1, P2 and P1+P2. Six haplotypes (TTAA, TTGG, TTAG, CTAA, CTGG, and CTAG) were inferred based on significant loci (AX-75730546 and AX-76530282) also showed significant association with fertility rate, where haplotype CTAG was shown to be markedly associated with the significantly highest (
P
< 0.0001) fertility rate (in P1, 86.42 ± 0.59; P2, 85.98 ± 0.59 and P1+P2, 86.16 ± 0.42) followed by other haplotypes for the irrespective of population studied. Collectively, we report for the first time that 12 SNP in the chromosomes 3 and 13 were significantly associated with fertility rate during the later stage of egg production, which could be used as the potential genetic markers that would be able to facilitate in the selection and improvement of fertility rate through chicken breeding.