Abstract <p>The SQUAMOSA Promoter-Binding Protein-Like (SPL) gene family plays critical roles in plant development, regulatory pathways, and stress adaptation. This study presents a comprehensive genome-wide analysis of SPL genes across four key Cucurbitaceae species -<i>Cucurbita maxima</i> (pumpkin), <i>Cucumis melo</i> (melon), <i>Cucumis sativus</i> (cucumber), and <i>Momordica charantia</i> (bitter gourd). We identified a diverse set of SPL genes: 27 in <i>C. maxima</i>, 21 in <i>C. melo</i>, 20 in <i>C. sativus</i>, and 16 in <i>M. charantia</i>, characterized by structural and functional diversity. Detailed analysis revealed significant variations in molecular weights, amino acid sequences, and subcellular localization patterns. Phylogenetic analysis classified SPL genes into distinct evolutionary groups, indicating functional conservation and divergence among these species. Functional predictions, supported by comparative analysis with model plants, particularly Arabidopsis thaliana, suggested potential roles in flowering regulation, leaf morphogenesis, and abiotic stress responses. The miR156-mediated regulatory network was also analyzed, identifying target sites within SPL genes and revealing intricate post-transcriptional regulation across different tissues and developmental stages. The study also uncovered both conserved and species-specific regulatory patterns, highlighting adaptive strategies employed by each species. Although primarily a bioinformatic study, the findings underscore the critical functions of SPL genes in growth, stress tolerance, and crop improvement. These results provide a foundation for future experimental validation, including gene knockout and overexpression studies, aimed at enhancing crop resilience and productivity through genetic interventions. Overall, this research contributes to sustainable agricultural practices and global food security.</p> Graphical abstract <p></p>