The Acanthaceaeis an important family of plants and it is also known as acanthus family, comprised of 250 genera and 4000 species spread globally in subtropical and tropical areas. From various geographical areas of Pakistan twelve species of Acanthaceae were collected to study its phylogeny utilizing ribosomal protein subunit 14 (rps14) gene. CTAB method was followed for the extraction of DNA from the leaves of collected plant samples. Primer was designed by using an online software primer3 (version 4.0). The sequence analysis was done using molecular evolutionary genetic analysis (MEGA 7), Iterative Threading Assembly Refinement (I-TASSER) and The Structure Analysis and Verification Server (SAVES). Our results showed two main clades of all twelve species revealing low genetic diversity (0.01) based on well supported bootstrap (BS) values. Furthermore, the pairwise distances ranged from 0.027 to 0.130 with mean value 0.078 was observed. To determine the 3-D protein structure and function of rps14 amino acid sequences, stereochemical analysis was performed using Ramachandran plots and I-TASSER. 3-D protein modeling depicted that rps14 protein from ten species out of twelve have the best quality structure with ≤ 2.0 % residues in disallowed region. Both phylogenetic analysis (amino acid and nucleotide sequences) revealed monophyletic origin of all species. Based on these results future studies should focus on physiological, taxonomical and molecular characters of family Acanthaceae species to determine lineages of all taxa within this diverse family.