Cotton (Gossypium hirsutum L.) is the backbone of Pakistan’s economy and the primary source of natural fiber worldwide. Despite its significance, cotton productivity remains suboptimal due to a narrow genetic base, biotic and abiotic stresses, and declining soil fertility. Breeding programs therefore focus on developing new, high-yielding, and stress-tolerant advance lines that can outperform existing commercial cultivars. In this study, field evaluation and molecular profiling were conducted on a set of advance lines derived from FH-490 and compared with standard commercial checks (e.g., FH-942, SLH-2010, CIM-70). The experiment was laid out in a randomized complete block design with three replications under normal sowing conditions. Agronomic traits including plant height, monopodial and sympodial branches, number of bolls, boll weight, and seed cotton yield were recorded alongside fiber quality and physiological parameters. Significant variation was observed among the genotypes, with L-1 and L-4 recording the highest yields, while FH-942 and SLH-2010 performed poorly. Molecular analysis using SSR markers revealed polymorphism that differentiated the advance lines, with SSR4-170 notably associated with tolerance under limited water conditions. The integration of field performance with DNA fingerprinting allowed a clearer understanding of both genetic diversity and adaptive potential. This study identifies promising lines (L-1, L-2, L-4, L-5) with superior yield and fiber quality, suggesting their suitability for inclusion in breeding pipelines. Findings emphasize the importance of combining morphological evaluation with molecular tools to accelerate the development of resilient cotton varieties capable of sustaining production under evolving climatic and resource constraints.