Abstract
Class III peroxidases, a class of antioxidant enzymes that regulate plant growth and development, contribute significantly to abiotic stress tolerance. However, comprehensive studies of the Class III PRX gene family in alfalfa (Medicago sativa) remain limited. Here, we identified and characterized the PRX gene family in alfalfa using bioinformatics approaches based on the Medicago sativa L. cv. ‘Zhongmu No.1’ reference genome. In total, 79 MsPRX genes were identified, which were unevenly distributed across the eight chromosomes. Furthermore, these genes were classified into 13 subgroups, and members of each subgroup exhibited conserved gene structures and motif distributions. Moreover, promoter cis-element analysis indicated the presence of numerous hormone-responsive and abiotic stress-responsive elements in promoter regions of the MsPRXs. Additionally, expression analysis revealed tissue-specific patterns, with higher expression levels in aboveground tissues than in belowground. Several MsPRX genes were strongly induced by low temperature, high temperature, drought, salt, and ABA treatments, suggesting their critical roles in abiotic stress regulation. This study systematically examines the identification, classification, evolution, and functional roles of PRXs in alfalfa. Consequently, these findings provide valuable insights into the evolutionary history and molecular mechanisms of PRXs, and foundations for future functional studies and genetic improvement of alfalfa. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12870-025-07325-9.