Lignans are critical bioactive compounds in sesame (Sesamum indicum L.), valued for their health benefits and industrial potential.  However, the genetic mechanisms underlying the lignan biosynthesis pathway remain poorly understood.  This study aimed to identify key quantitative trait loci (QTLs) associated with lignan content using 140 F₉ recombinant inbred lines (RILs) derived from a cross between the sesame cultivars Areum and Gomazou, and to develop molecular markers for breeding programs to enhance lignan content.  Genotyping-by-sequencing (GBS) technology was employed, combining single (ApeKI) and double-enzyme (PstI/MspI) digestions.  This approach provided comprehensive genome coverage and substantially improved the quality of the linkage map.  A total of 19 QTLs associated with lignan components, including oil-soluble and water-soluble lignans, were identified.  Among these, loci on chromosomes 6 and 11 exhibited high LOD scores of 17.8 (PVE 13.9%) and 51.5 (PVE 68.7%), respectively, for total lignan contents in seeds.  Two major QTLs, qLIGNAN6 and qLIGNAN11, were identified as key contributors to lignan content.  Significant single nucleotide polymorphisms (SNPs) linked to these QTLs were used to develop CAPS markers for potential application in marker-assisted selection (MAS).  These markers were validated in RILs and germplasm to evaluate beneficial allele stacking and gene pyramiding.  This study provides valuable genetic resources and molecular markers to facilitate the effective implementation of sesame breeding strategies to improve nutritional value.  The findings contribute to the development of sesame varieties with higher lignan content, supporting both health and industrial applications.  

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