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Aim The aim of this study was to investigate the SARS-CoV-2 spike protein evolution during the first and second wave of COVID-19 infections in India. Materials Methods Detailed mutation analysis was done in 763 samples taken from GISAID for the ten most affected Indian states between March 2020 to August 2021. Results The study revealed 242 mutations corresponding to 207 sites. Fifty one novel mutations emerged during the assessment period, including many with higher transmissibility and immune evasion functions. Highest number of m