Abstract
Cotton production worldwide is severely threatened by soil‐borne fungal pathogens, primarily Verticillium dahliae (VD) and Fusarium oxysporum f . sp. vasinfectum (FOV). These wilts can cause seedling loss and substantial yield reduction, making the development of resilient cultivars essential. This study utilized an integrated breeding approach, combining marker‐assisted selection (MAS) with rigorous phytopathological and biochemical analyses, to accelerate the development of wilt‐resistant Gossypium hirsutum lines. We screened 10 fungal isolates and 8 complex interlinear hybrids across multiple generations (F 7 –F 11 ). Genotyping using six SSR markers, including BNL4003, CM209, and TMB0161, identified the hybrid CBRI_3 as the most promising source of resistance alleles. Biochemical studies confirmed that two hybrids, CBRI_3 and CBRI_8, exhibited superior resistance. In these resistant lines, peroxidase enzyme activity was significantly 1.3–1.6 times higher than that in susceptible controls, promoting vital cell wall reinforcement and lignification, which are key defenses against fungal penetration. This integrated approach successfully led to the isolation of the highly resistant line, L‐1077 (derived from CBRI_3). This line maintains high fiber quality (Type IV standard) and superior yield while showing strong resistance to both V. dahliae and FOV. The line L‐1077 was submitted for patent registration under the variety designation C‐6602 (NAP 2020 0007).