Lipoxygenases (LOXs; EC1.13.11.12) are a family of iron- or manganese-containing dioxygenases that catalyze the oxygenation of polyunsaturated fatty acids (PUFAs). In plants, these enzymes are encoded by a multigene family and play key roles in growth, development, and responses to biotic and abiotic stresses. This study aimed to determine the evolutionary characteristics and members of the LOX gene family in Medicago truncatula (annual medicago) and their function in stress responses. The LOX gene family in M. truncatula was identified, and their general characteristics, phylogenetic relationships, gene structure, conserved domains, cis-regulatory elements in the promoter region, post-transcriptional regulation, and expression were studied. Twenty-five homologous LOX genes were identified and classified into 9-LOX, 13-LOX type I, and 13-LOX type II subgroups based on phylogeny with chickpea, bean, soybean, rice, Arabidopsis, and maize. The LOX genes contained conserved lipoxygenase and PLAT/LH2 domains. Conservation was also observed in gene structure, intron phase, and physicochemical properties of the encoded proteins. The presence of cis-regulatory elements responsive to hormones and stresses (e.g., ERE, MYB, MYC) in LOX promoters suggests their roles in plant development and environmental stress responses. Additionally, various identified miRNAs regulate post-transcriptional expression of LOX genes via cleavage or translational inhibition. This study provides valuable information for understanding LOX gene functions and further exploration of the LOX gene family in M. truncatula.