This investigation involved a comparative analysis of the small GTPase superfamily in S. lycopersicum super strain B compared to their analogues in leguminous and other non-leguminous species. The small GTPases superfamily members were recognized by tBLASTn searches. The sequences of amino acid were aligned using Clustal Omega and the analysis of phylogeny was performed with the MEGA7 package. Protein alignments were applied for all studied species. Three-dimensional models of RABA2, ROP9, and ROP10 from Solanum lycopersicum “Super strain B” were performed. The levels of mRNA of the Rab, Arf, Rop, and Ran subfamilies were detected in aerial tissues vs. roots. Significant divergences were found in the number of members and groups comprising each subfamily of the small GTPases and Glycine max had the highest count. High expression of Rab and Arf proteins was shown in the roots of legumes whilst in non-legume plants, the highest values were recorded in aerial tissues. S. lycopersicum super strain B had the highest expression of Rab and Arf proteins in its aerial tissues, which may indicate that diazotroph strains have supreme activities in the aerial tissues of strain B and act as associated N-fixing bacteria. The phylogenies of the small GTPase superfamily of the studied plants did not reveal asymmetric evolution of the Ra, Arf, Rop, and Ran subfamilies. Multiple sequence alignments derived from each of the Rab, Arf, and Rop proteins of S. lycopersicum super strain B showed a low frequency of substitutions in their domains. GTPases superfamily members have definite functions during infection, delivery, and maintenance of N2-fixing diazotroph but show some alterations in their function among S. lycopersicum super strain B, and other species.