In light of the emergence of new and potentially more virulent SARS-CoV-2 variants, an in-depth analysis of the main protease (Mpro) or 3CLpro, a critical target for COVID-19 therapy due to its essential role in viral replication, was conducted. This study focused on the alpha, beta, gamma, delta, and omicron variants, comparing them to the reference sequence (Wuhan-Hu-1). The comprehensive analysis of the mutational landscape of SARS-CoV-2 Mpro across Variants of Concern (VOCs) provides valuable insights into the evolving nature of the virus. The study utilized a substantial dataset of 222,980 sequences (quality data) and created a library of a total of 51,733 mutations for the Mpro protein. The distribution of mutations varied among the variants, with distinct patterns observed. Certain regions crucial for drug binding and protease function showed relatively fewer mutation rates, unlike the interdomain site, which exhibited higher mutation rates compared to catalytic and dimeric interface sites. Notably, the Omicron variant displayed the prevalent P132H mutation, while the Beta variant featured the prevalent K90R mutation. Furthermore, proline positions in Mpro were investigated, with mutations identified in ten out of thirteen positions, underscoring the potential impact on protein stability. Additionally, the catalytic site S1 sub-pocket was found to be less mutated, whereas the S3 sub-pocket was highly mutated. These findings suggest a critical role in maintaining the structural and functional integrity of Mpro, offering valuable insights for future research and therapeutic development.