The thermolysin family of enzymes is classified as the M4 family of metallopeptidases. M4 family comprises numerous zinc-dependent metallopeptidases that hydrolyze peptide bonds. Zinc containing peptidases are widely distributed in nature and have important roles in many physiological processes. M4 family comprises numerous zinc-dependent metallopeptidases that hydrolyse peptide bonds. Metallopeptidases were studied for the reason of their great relevance to biology, medicine and biotechnology. In the present study, detailed comparative analyses of both thermophilic and psychrophilic metallopeptidases were performed. The comparative analysis of both thermophilic and psychrophilic metallopeptidases was performed by taking sequence parameters such as amino acid composition, amino acid property group composition, physico-chemical properties, secondary structure content. From comparison between the two groups, it was found that Gln, Asn, Ser, Thr, and His are significantly lower in thermophilic metallopeptidase. Positively charged residues (Lys, Arg and Glu) are more significant in thermophilic metallopeptidases than in psychrophilic metallopeptidases. By Scan Prosite tool it was found that VSAHEVSHGF and VIGHETHAV or VVGHETHV amino acid pattern were common in all psychrophilic and thermophilic metallopeptidases respectively. By studying the secondary structure, it was found that the content of helices is more in thermophilic metallopeptidase and strand is overrepresented in psychrophilic metalopeptidases. These findings provide the required knowledge that these factors are useful for differentiating the thermophilic and psychrophilic metallopeptidases and are helpful for comparative analysis of thermophilic and psychrophilic metallopeptidases.