Members of the PIP4K/PIP5K family of proteins, which generate the highly important secondary messenger phosphatidylinositol-4,5-bisphosphate, play central roles in regulating diverse signaling pathways. In eukaryotic organisms, multiple isozymes and subfamilies of PIP4K/PIP5K proteins are found and it is of much interest to understand their evolution and species distribution and what unique molecular and biochemical characteristics distinguish specific isozymes and subfamilies of proteins. We report here the species distribution of different PIP4K/PIP5K family of proteins in eukaryotic organisms and phylogenetic analysis based on their protein sequences. Our results indicate that the distinct homologs of both PIP4K and PIP5K are found in different organisms belonging to the Holozoa clade of eukaryotes, which comprises of various metazoan phyla as well as their close unicellular relatives Choanoflagellates and Filasterea. In contrast, the deeper-branching eukaryotic lineages, as well as plants and fungi, contain only a single homolog of the PIP4K/PIP5K proteins. In parallel, our comparative analyses of PIP4K/PIP5K protein sequences have identified six highly-specific molecular markers consisting of conserved signature indels (CSIs) that are uniquely shared by either the PIP4K or PIP5K proteins, or both, or specific subfamilies of these proteins. Of these molecular markers, 2 CSIs are distinctive characteristics of all PIP4K homologs, 1 CSI distinguishes the PIP4K and PIP5K homologs from the Holozoa clade of species from the ancestral form of PIP4K/PIP5K found in deeper-branching eukaryotic lineages. The remaining three CSIs are specific for the PIP5Kα, PIP5Kβ, and PIP4Kγ subfamilies of proteins from vertebrate species. These molecular markers provide important means for distinguishing different PIP4K/PIP5K isozymes as well as some of their subfamilies. In addition, the distribution patterns of these markers in different isozymes provide important insights into the evolutionary divergence of PIP4K/PIP5K proteins. Our results support the view that the Holozoa clade of eukaryotic organisms shared a common ancestor exclusive of the other eukaryotic lineages and that the initial gene duplication event leading to the divergence of distinct types of PIP4K and PIP5K homologs occurred in a common ancestor of this clade. Based on the results gleaned from different studies presented here, a model for the evolutionary divergence of the PIP4K/PIP5K family of proteins is presented.