Abstract: Both small GTPase and its activating protein (GAP) superfamilies exist in various eukaryotes. The small GTPases regulate a wide variety of cellular processes by cycling between active GTP- and inactive GAP-bound conformations. The GAPs promote GTPase inactivation by stimulating the GTP hydrolysis. In this study, we identified 111 small GTPases and 85 GAPs in rice, 65 GAPs in Arabidopsis, 90 small GTPases in D. melanogaster, and 35 GAPs in S. cerevisiae by genome-wide analysis. We then analyzed and compared a total of 498 small GTPases and 422 GAPs from these four eukaryotic and human genomes. Both animals and yeast genomes contained 5 families of small GTPases and their GAPs. However, plants had only 4 of these 5 families due to a lack of the Ras and RasGAP genes. Small GTPases were conserved with common motifs but GAPs exhibited higher and much more rapid divergence. Based on phylogenetic analysis of all small GTPases and GAPs in 5 eukaryotic organisms, we estimated that their ancestors had small sizes of small GTPases and GAPs and their large-scale expansions occurred after the divergence from their ancestors. Further investigation showed that genome duplications represented the major mechanism for such expansions. Non-synonymous substitutions per site (Ka) and synonymous substitutions per site (Ks) analysis showed that most of the divergence due to a positive selection occurred in common ancestors, suggesting a major functional divergence in an ancient era.