BACKGROUND: COVID-19 pandemic had unprecedented global impact on health and society, highlighting the need for a detailed understanding of SARS-CoV-2 evolution in response to host and environmental factors. This study investigates the evolution of SARS-CoV-2 via mutation dynamics, focusing on distinct age cohorts, geographical location, and vaccination status within the Indian population, one of the nations most affected by COVID-19. METHODOLOGY: Comprehensive dataset, across diverse time points during the Alpha, Delta, and Omicron variant waves, captured essential phases of the pandemic's footprint in India. By leveraging genomic data from Global Initiative on Sharing Avian Influenza Data (GISAID), we examined the substitution mutation landscape of SARS-CoV-2 in three demographic segments: children (1-17 years), working-age adults (18-64 years), and elderly individuals (65+ years). A balanced dataset of 69,975 samples was used for the study, comprising 23,325 samples from each group. This design ensured high statistical power, as confirmed by power analysis. We employed bioinformatics and statistical analyses, to explore genetic diversity patterns and substitution frequencies across the age groups. PRINCIPAL FINDINGS: The working-age group exhibited a notably high frequency of unique substitutions, suggesting that immune pressures within highly interactive populations may accelerate viral adaptation. Geographic analysis emphasizes notable regional variation in substitution rates, potentially driven by population density and local transmission dynamics, while regions with more homogeneous strain circulation show relatively lower substitution rates. The analysis also revealed a significant surge in unique substitutions across all age groups during the vaccination period, with substitution rates remaining elevated even after widespread vaccination, compared to pre-vaccination levels. This trend supports the virus's adaptive response to heightened immune pressures from vaccination, as observed through the increased prevalence of substitutions in important regions of SARS-CoV-2 genome like ORF1ab and Spike, potentially contributing to immune escape and transmissibility. CONCLUSION: Our findings affirm the importance of continuous surveillance on viral evolution, particularly in countries with high transmission rates. This research provides insights for anticipating future viral outbreaks and refining pandemic preparedness strategies, thus enhancing our capacity for proactive global health responses.