Crop productivity is declining due to climate change, with sesame being particularly vulnerable due to its narrow genetic base. To address this, 28 advanced sesame lines were evaluated across 23 environments in Egypt over six growing seasons (2018–2023). The study aimed to identify stable and high-yielding genotypes for developing climate-ready cultivars. Additive main effects and multiplicative interaction analyses were employed to assess genotype stability and performance. Molecular markers (RAPD and ISSR) were used to characterize genetic variation and identify markers linked to yield and stability. The experiments were performed in a randomized complete block design (RCBD) with three replicates. The results showed that the genotypes had significant differences (p < 0.05) for seed yield. AMMI analysis identified C5.8, C3.8, C6.11, C6.2, and C9.6 as stable and high-yielding genotypes. RAPD (random amplified polymorphic DNA) and ISSR (inter-simple sequence repeat) markers revealed high levels of genetic variation; eight RAPD and ten ISSR primers amplified 78 and 84 bands with a mean polymorphism of 77.5% and 60.47%, respectively. In addition to seven markers associated with yield and stability. These findings highlight the potential of these advanced lines for developing climate-ready cultivars.