Background: This study aims to comprehensively analyze the phosphatidylinositol-specific phospholipase C (PI-PLC) gene family in Leishmania infantum and Leishmania major genomes. By elucidating the roles of PI-PLC enzymes in parasite biology and their potential as therapeutic targets, the research contributes to understanding molecular mechanisms underlying parasite proliferation and pathogenesis. Methods: Genomic screening, gene structure analysis, phylogenetics, chromosomal distribution, and expression profiling of PI-PLC genes in L. infantum and L. major were conducted. RNA-seq data from susceptible and resistant L. infantum strains to antimony treatment were analyzed to assess gene expression patterns. Molecular docking and molecular dynamics simulations explored Evodone’s interaction within the active sites of proteins 2B4W and 4TYZ. Results: A total of 22 PI-PLC genes were identified in both L. infantum and L. major genomes, characterized by conserved domains and biochemical properties. Phylogenetic analysis revealed evolutionary relationships and clustering patterns of these genes. Chromosomal distribution and expression profiling provided insights into their functional diversity and potential involvement in drug resistance mechanisms. Conclusion: This study highlights the significant roles of PI-PLC enzymes in Leishmania biology and their potential as targets for novel therapeutic interventions. Understanding their involvement in parasite pathogenesis and drug resistance mechanisms could pave the way for developing effective treatments against leishmaniasis.