ABSTRACT Vitamin A deficiency (VAD), a major global health concern, has driven efforts to develop staple crops with enhanced pro‐vitamin A (pVA) content. Delivering meaningful nutritional benefits, however, requires technologies that maintain elevated carotenoid levels under field conditions. Previous proof‐of‐concept work demonstrated that pVA content can be substantially increased in Cavendish bananas through genetic modification, providing a platform for transferring the technology into East African Highland banana (EAHB) cultivars relevant to reducing VAD in Uganda. To evaluate performance under agronomic conditions, we conducted multi‐generational field assessments of 27 transgenic Cavendish lines generated from seven constructs enabling constitutive or fruit‐preferred expression of three carotenoid biosynthesis genes: ZmPsy1 , MtPsy2a and PaCrtI . Constitutive expression was driven by the maize Ubi promoter, while fruit expression was regulated by Exp1 or ACO promoters. Agronomic performance and fruit carotenoid levels were analysed across three generations to explore factors influencing pVA enhancement. All transgenic lines exhibited increased fruit pVA, with the highest accumulation observed in lines constitutively expressing MtPsy2a . Promoter‐transgene combinations significantly affected carotenoid accumulation and the stability of the trait in the field. PVA accumulation was the highest in the initial sucker crop and declined in subsequent ratoons, reflecting sensitivity to seasonal conditions. While ACO‐ and Ubi‐driven lines were less affected by seasonal temperature changes, these variations significantly constrained pVA accumulation in wild‐type and Exp1‐regulated lines. This comprehensive assessment helps elucidate the complex interplay of promoter, isoform, and environmental factors that are essential for the long‐term viability of nutritional interventions aimed at combating VAD in the region.