Ginger ( Zingiber officinale Rosc.) holds significant culinary and medicinal value. Tissue-cultured ginger can markedly enhance rhizome yield after two years of growth. This study investigates the field performance variations and reveals the influence mechanism of root on plant morphology and yield among different ginger generations. The results indicate that the T1, T2 and T3 gingers exhibit strong genetic stability. Compared to T1 and T2 gingers, T3 ginger show superior performance in both aboveground and belowground biomass, photosynthetic capacity, yield and quality. However, anatomical analysis reveals that T3 ginger has a smaller diameter for fibrous roots and a lower cortex-to-stele thickness ratio in the fleshy roots compared to T1 and T2 gingers. Additionally, transcriptomic analysis elucidates the relationship between root development and metabolic regulation across different ginger generations. Compared to T2 roots, T3 roots exhibit significantly higher expression levels of DEGs associated with starch and sucrose metabolism, along with increased activity of starch-degrading enzymes (BAM), which ultimately influence the accumulation and conversion of starch and sucrose. Meanwhile, key enzymes activity and DEGs expression in flavonoid biosynthesis were downregulated, resulting in a significant reduction of flavonoid content in T3 roots. Furthermore, levels of ABA, TZR and SA were markedly lower in T3 roots, whereas the concentrations of IAA, JA and GA 3 were significantly higher. In conclusion, this study reveals notable differences in morphology, root development and rhizome yield across various generations of ginger. Specifically, T3 ginger exhibit superior yield and quality with weaker root development, which may be related to the dynamic regulations of starch and sucrose metabolism, flavonoid biosynthesis and phytohormone levels. These findings provide valuable theoretical insights and practical recommendations for optimizing the field cultivation of tissue-cultured ginger.