Tetranychus urticae, a globally ubiquitous mite, poses a significant threat to agriculture. Elevated temperatures exacerbate the growth, development, and reproduction of T. urticae, leading to substantial crop damage. In this study, we employed comparative transcriptomic approaches with whole-genome information of T. urticae to identify six Glutathione S-transferase genes (GSTs) implicated in heat stress response. Through comprehensive bioinformatics analyses, we elucidated the tertiary structure and active sites of the corresponding proteins, providing a thorough characterization of these GST genes. Furthermore, we investigated the expression patterns of these six GST genes under short-term heat shock conditions. Our findings unveiled the involvement of T. urticae GST genes in combating oxidative stress induced by heat, underscoring their role in antioxidant defense mechanisms. This study contributes valuable insights into the molecular mechanisms underlying the response of T. urticae to heat stress, laying a foundation for the development of strategies aimed at mitigating its impact in high-temperature environments.