Increasing levels of UV-B radiation caused by the greenhouse effect has become an emerging threat to crop health and yield. The glutathione (GSH) metabolic pathway is generally involved in plant stress responses through scavenging accumulated reactive oxygen species, and is therefore believed to play an essential role in enhancing plant tolerance to UV-B stress. However, the complex evolutionary details of this pathway in polyploid plants, especially under UV-B stress, remain largely unknown. Here, using the important allotetraploid crop, Gossypium hirsutum, as an example, we comprehensively investigated the composition and phylogenetic relationships of genes encoding 12 key structural enzymes in this pathway, and compared the expression changes of all the relevant genes under UV-B stress (16 kJ m-2 d-1) based on six leaf transcriptomes. Consequently, we identified 205 structural genes by genome-wide searching and predicted 98 potential regulatory genes under multiple stress conditions by co-expression network analysis. Furthermore, we revealed that 19 structural genes including five homoeologous pairs and 96 regulatory genes possessing 25 homoeologous pairs were reticulately correlated without homoeologous selection preference under UV-B stress. This result suggests a complex rewiring and reassignment between structural genes and their regulatory networks in the duplicated metabolic pathways of polyploid cotton. This study extends our understanding of the molecular dynamics of the GSH metabolic pathway in response to UV-B stress in G. hirsutum and, more broadly, in polyploid plants.