Oxidative stress reflects an imbalance between the systemic manifestation of reactive oxygen species (ROS) and a biological system's ability to readily detoxify the reactive intermediates or to repair the resulting damage. Glutaredoxins (GRXs) are ubiquitous oxidoreductase enzymes involved in diverse cellular processes and play a key role in oxidative stress responsive mechanisms. This study was aimed to explore the structure-function relationship and to provide a framework for functional validation and biochemical characterization of various GRX members. In this study, our analysis revealed the presence of 127 genes encoding GRX proteins in G. hirsutum. A total of 758 genes from two typical monocot and nine dicot species were naturally divided into four classes based on phylogenetic analysis. The classification was supported with organization of conserved protein motifs and sequence logos comparison between cotton, rice and Arabidopsis. Cotton GRX gene family has underwent strong purifying selection with limited functional divergence. A good collinearity was observed in the synteny analysis of four Gossypium species. Majority of cotton GRXs were influenced by various phytohormones and abiotic stress conditions during expression analysis, suggesting an important role of GRX proteins in response to oxidative stress. Cis-regulatory elements, gene enrichments and co-expression network analysis also support their predicted role against various abiotic stresses. Whole genome and segmental duplication were determined to be the two major impetuses for the expansion of gene numbers during the evolution. The identification of GRX genes showing differential expression in specific tissues or in response to environmental stimuli provides a new avenue for in-depth characterization of selected genes of importance. This study will further broaden our insights into the evolution and functional elucidation of GRX gene family in cotton.