Objective To identify key amino acid variations of major proteins from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by biophysical methods. Methods Through amino acid sequence alignment, classification of variant amino acid residues, three-dimensional structure reconstruction of proteins, and electrostatic interaction analysis of amino acid residues, the key amino acid variations of major proteins from SARS-CoV-2 was analyzed with RaTG13, the bat coronavirus with the highest homology, as the reference. Results At least ten amino acid variations that affect the possible electrostatic interactions were identified in RNA-dependent RNA polymerase (RdRp), exoribonuclease (ExoN), uridylate-specific endoribonuclease (NendoU), and spike (S) protein from SARS-CoV-2. These variations may affect the spatial conformation and biological functions of the proteins. Conclusion The key amino acid variations of the major proteins from SARS-CoV-2 have been preliminarily identified, providing clues for understanding the genetic, pathogenic and epidemiological characteristics of the virus.