This study focused on identifying and analyzing the EDS1 genes in cucumber using various bioinformatics tools and databases. The EDS1 (the ENHANCED DISEASE SUSCEPTIBILITY 1) and PAD4 (PHYTOALEXIN DEFICIENT 4) proteins play a significant role in regulating gene expression in response to biotic and abiotic stresses. The study identified three CsEDS1 genes in cucumber through BLAST-p analysis and analyzed their gene structures, conserved motifs, and domains. The phylogenetic analysis was used to determine the evolutionary relationship between CsEDS1 and other related species. Cisregulatory element analysis was performed to evaluate the putative function of various cis-elements in genes, and transcriptome analysis under different conditions showed the expression patterns of CsEDS1 genes. Subcellular localization analysis showed that all CsEDS1 sequences were localized in the nucleus, cytoplasm, and chloroplast. The study used Ks/Ka analysis to reveal that purification selection by the environment was the result of the evolution of CsEDS1 genes. Chromosomal mapping and synteny analysis showed segmental duplication of CsEDS1 genes. These results provide valuable insights into the evolution and function of CsEDS1 genes in cucumber, highlighting their significant role in plant survival and adaptation to different stress conditions. The findings of this study have implications for crop improvement and stress tolerance in plants. Understanding the molecular characteristics and evolutionary relationships of CsEDS1 genes can help develop strategies for improving plant resistance to biotic and abiotic stresses. The study also sheds light on the function of EDS1 and PAD4 proteins in regulating gene expression in response to environmental stresses, contributing to basal immunity through transcriptional regulation, and demonstrating different modes of action and pathway connectivities.