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Simple Summary: The climatic changes pose important threats to agriculture. Plants are sessile organisms and need to cope with different environmental conditions throughout their lifespan. Most responses involve different signaling molecules, including reactive oxygen species (ROS). Due to their high reactivity, ROS are also an oxidative threat to the cell. Consequently, plant cells display elaborated defenses against oxidative stress. The ascorbate-glutathione cycle is the main antioxidant pathway in photosynthetic organisms and is composed by the enzymes ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR). Here, the APX, MDAR, DHAR and GR genes from castor bean, cassava, jatropha and rubber tree were identified and classified. This classification allowed the prediction of their subcellular localization within plant cells, such as cytosol, peroxisomes, chloroplasts, and mitochondria. Our analysis also contributes to understanding the evolutionary history of these genes. The expression pattern of ascorbate-glutathione cycle genes in castor bean submitted to drought reveals changes in leaves and roots. Altogether, these data contribute to uncovering the regulation of ROS metabolism during stress response in castor bean, which is highly tolerant to drought. Ascorbate peroxidase (APX), Monodehydroascorbate Reductase (MDAR), Dehydroascorbate Reductase (DHAR) and Glutathione Reductase (GR) enzymes participate in the ascorbate-glutathione cycle, which exerts a central role in the antioxidant metabolism in plants. Despite the importance of this antioxidant system in different signal transduction networks related to development and response to environmental stresses, the pathway has not yet been comprehensively characterized in many crop plants. Among different eudicotyledons, the Euphorbiaceae family is particularly diverse with some species highly tolerant to drought. Here the APX, MDAR, DHAR, and GR genes in Ricinus communis, Jatropha curcas, Manihot esculenta, and Hevea brasiliensis were identified and characterized. The comprehensive phylogenetic and genomic analyses allowed the classification of the genes into different classes, equivalent to cytosolic, peroxisomal, chloroplastic, and mitochondrial enzymes, and revealed the duplication events that contribute to the expansion of these families within plant genomes. Due to the high drought stress tolerance of Ricinus communis, the expression patterns of ascorbate-glutathione cycle genes in response to drought were also analyzed in leaves and roots, indicating a differential expression during the stress. Altogether, these data contributed to the characterization of the expression pattern and evolutionary analysis of these genes, filling the gap in the proposed functions of core components of the antioxidant mechanism during stress response in an economically relevant group of plants. [ABSTRACT FROM AUTHOR]