Malpighiales, containing approximately 16,000 species, is one of the largest and most diverse angiosperm orders in plants. Growth-regulating factor (GRF) protein is a putative transcription factor and plays a regulatory role during plant growth and development processes such as stem elongation and cell expansion. The latest available protein data provide an opportunity to compare and understand the critical similarities and differentiation in GRF proteins among Malpighiales members. In the present study we conducted domain, two- and three-dimensional (2D, 3D) comparative, physicochemical, subcellular prediction, and sequence analysis of 87 putative GRF proteins belonging to 4 genera in Malpighiales using different bioinformatic tools. The results showed that QLQ, WRC, TQL, GGPL, RSRKPVE, FDV, and GFG domain motifs are conserved in rates between 34% and 82%. Comparative analysis proved that there is a common triangle-like shape among GRF proteins, and the conserved motifs are localized in similar regions of this triangle. The sequence lengths of GRFs change between 93 and 639 aa, and pH values differ between 5.28 and 10.50. According to subcellular prediction and domain analysis, only 9%-11% of GRFs are localized out of the nuclear region. Additionally, it was determined that 87 GRF proteins have monophyletic phylogeny. The average values of pairwise distance and nucleotide diversity (p) were 2.78 and 0.727, respectively. These results may contribute to an understanding of how structurally similar but functionally diverse GRF proteins execute various functions in different plant parts or in different plant species.