A previous genome-wide transcriptional analysis identified long non-coding RNA 8138.1 (lncRNA8138.1) as a candidate gene related to hen duration of the fertility (DF) trait. LncRNA8138.1 gene response to growth factor and reproductive system development suggests it has a vital role in reproduction. In this study, we inves- tigated the lncRNA8138.1 gene sequence in a population of egg-laying hens. The sequence analysis of the lncRNA8138.1 gene containing about 1.6 k nucleotides (nt) was observed with four single nucleotide polymorphisms (SNPs) and 7 nt indel including r.4937159A > G; r.4937219T > C; r.4937258G > C; r.4937318C > G and g.4937319_4937325delinsTGTGTGG. Next, the genomic DNAs from laying hen populations were subjected to polymerase chain reaction (PCR) and restric- tion fragment length polymorphism (RFLP) to detect a region of 457 bp carrying lncRNA8138.1 r.4937159A >Gsubstitution. Further inspection of the region containing r.4937159A > G mutation revealed three genotypes viz., AA, AG, and GG were observed with respective frequencies of 0.106, 0.607, and 0.287 in laying hen population 1 (P1) (n D 1;042) and respective frequencies of 0.176, 0.708, and 0.116 in laying hen population 2 (P2) (n D 826). Moreover, to further examining the frequencies of r.4937159A > G genotypes in P1 and P2, and their additive and dominance effects; r.4937159A > G locus was significantly associated with DF-trait in both P1 and P2 (EN: the number of eggs, FN: the number of fertile eggs after a single AI), and DN (the number of days post-insemination until last fertile egg). In testing for additive and dominance effects, additive effect was significant (P G polymorphism is a potential biomarker for DF-trait. However, the identified novel r.4937159A > G mutation and others require further investigation to confirm phenotypic causality and potential genetic relationships with reproductive traits. Overall, our findings suggest the significance of genetic variation in long non-coding RNAs may assist in future breeding programs to improve selection for prolonged DF-trait.