Thermostable proteases are one of the pivotal enzymatic groups which play fundamental roles in biotechnologyrelated industries. The identification of bacterial thermostable enzymes through screening programs is a time and cost consuming process. So, extensive bioinformatics and experimental studies have been conducted to reveal thermo stabilizing factors. The current study was aimed to evaluate distinctive indicators among 33 thermostable and 10 mesostable proteolytic enzymes. The frequency of individual amino acids, aliphatic indexes, melting temperatures, isoelectric points, as well as, the frequency of AXXXA and GXXXG motifs were determined and compared among these enzymes. In addition, types of proteolytic enzymes and their active sites were assigned. Moreover, the frequency of alpha helixes, polar surface regions, and packing volumes of these enzymes with the known structures were characterized. Results showed that the frequency of Ala and AXXXA motifs were significantly higher in thermostable proteolytic enzymes, while they possess lower contents of Met, His, Lys and Leu in comparison to mesostable enzymes (P<0.05). According to statistical analysis, thermostable proteolytic enzymes indicated meaningful lower packing volumes than mesostable enzymes (P<0.05). Findings of the current study in addition to more detailed investigations on the thermostability mechanisms of various protein families are essential for designing more efficient industrial enzymes with functional properties at high temperatures