Cowpea (Vigna unguiculata (L.) Walp.) is a major source of dietary protein in sub-Saharan Africa, but its productivity is severely constrained by the parasitic weed Striga gesnerioides. This study evaluated 140 BC₃F₂ cowpea lines developed through marker-assisted gene pyramiding for resistance to S. gesnerioides. Two segregating populations were assessed: Population 1 derived from {(IT90K-277-2 × B301) × (IT90K-277-2 × TVu-1272)} and Population 2 from {(IT90K-277-2 × TVu-16514) × (IT90K-277-2 × TVu-1272)}. The BC₃F₂ lines were screened in a replicated pot experiment using a mixed Striga seed inoculum collected from three major cowpea-producing areas in northern Nigeria. Analysis of variance revealed highly significant genetic variation (p < 0.0001) among lines for both agronomic traits and Striga resistance parameters. Broad-sense heritability estimates ranged from moderate to high, indicating strong genetic control of the evaluated traits. Hierarchical clustering and heatmap analyses identified a subset of BC₃F₂ lines combining low Striga infestation with favourable yield components, while strong negative correlations were observed between Striga infestation parameters and yield traits. Segregation analysis conformed to a two-gene dominant inheritance model for Striga resistance, with observed ratios fitting the expected 1:1:1:1 distribution (χ²= 2.34). The results demonstrate the effectiveness of marker-assisted pyramiding in combining resistance alleles from multiple donor parents and identify BC₃F₂ lines suitable for advancement towards the development of cowpea cultivars with stable resistance to S. gesnerioides in Striga-endemic production systems.