LuxI and LuxR are key factors that drive quorum sensing (QS) in bacteria through secretion and perception of the signaling molecules e.g. N-Acyl homoserine lactones (AHLs). The role of these proteins is well established in Gram-negative bacteria for intercellular communication but remain under-explored in Gram-positive bacteria where QS peptides are majorly responsible for cell-to-cell communication. Therefore, in the present study, we explored conservation, potential function, topological arrangements and evolutionarily aspects of these proteins in Gram-positive bacteria. Putative LuxI/LuxR containing proteins were retrieved using the domain-based strategy from InterPro v62.0 meta-database. Conservational analyses via multiple sequence alignment and domain showed that these are well conserved in Gram-positive bacteria and possess relatedness with Gram-negative bacteria. Further, Gene ontology and ligand-based functional annotation explain their active involvement in signal transduction mechanism via QS signaling molecules. Moreover, Phylogenetic analyses (LuxI, LuxR, LuxI + LuxR and 16s rRNA) revealed horizontal gene transfer events with significant statistical support among Gram-positive and Gram-negative bacteria. This in-silico study offers a detailed overview of potential LuxI/LuxR distribution in Gram-positive bacteria (mainly Firmicutes and Actinobacteria) and their functional role in QS. It would further help in understanding the extent of interspecies communications between Gram-positive and Gram-negative bacteria through QS signaling molecules.