Chemokines are chemotactic proteins involved in host defense through the migration of immune-regulatory cells to the site of infection. Interleukin-8 (CXCL8/IL8) is the most studied “ELR-CXC chemokine/neutrophil activating chemokine (NAC) that regulate neutrophil trafficking during infections and inflammation by binding to its cognate G-protein coupled receptors CXCR1/CXCR2. The “ELR” motif of NAC chemokines is essential for the CXCR1/CXCR2 receptor activation. In order to understand the evolutionary origin of “ELR” motif in the CXC chemokines, a thorough evolutionary study of CXCL8 gene from various fishes and primates was performed. Phylogenetic analysis revealed that the CXCL8 gene can be classified into four distinct lineages (CXCL8-L1a, CXCL8-L1b, CXCL8-L2, and CXCL8-L3), where CXCL8-L1a is the fastest evolving lineage and CXCL8-L3 is the slowest. Selection analysis suggested that The “ELR/DLR” motif containing branches (gadoid and coelacanth) are positively selected. The probable evolutionary trend of “ELR” motif suggested that this motif in ancestor CXCL8 is evolved from the GGR of Lamprey (Agnatha), followed by duplication giving rise to two main motifs in CXCL8 “NXH” in L3 lineage and “ELR/DLR” in L1a/L1b lineages. Although, structural analysis suggested that the overall topology of the CXCL8 proteins is similar, differences do exist at the individual structural elements among the members of different lineages. Functional distance analysis suggested that the CXCL8-L3 lineage is more distant compared to the CXCL8-L1a and L1b lineages from the inferred ancestor. Functional divergence analysis between different lineages suggested that most of the selected residues are important for receptor or glycosaminoglycan binding. Such a functional diversification can be attributed to the novel set of functions adopted by CXCL8 in various species. © 2019 Elsevier Ltd