The ongoing pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the virus's remarkable ability to evolve and adapt in diverse hosts. Despite the observation of recurrent mutations and convergent evolution in the viral genome, the mechanisms driving these processes remain poorly understood, particularly in the context of diverse host environments and limited genomic surveillance. We established a rigorously controlled in vitro cellular system within a Biosafety Level 3 Laboratory, ensuring strict adherence to biosafety protocols while passaging the virus in seven cell lines derived from four tissues across five mammalian species. High-throughput sequencing revealed consistent positive selection on the Spike (S) protein, highlighting its adaptability in the absence of adaptive immune responses or therapeutic pressures. Type I interferons (IFN-I) and APOBEC-mediated editing may emerge as key modulators of viral evolution. Notably, IFN-I activation is inversely correlated with the accumulation of S protein mutations (E484D, P812R/L, L1186R). Our findings uncover host-specific selective forces in shaping SARS-CoV-2 evolution and highlight the need for systematic approaches to mitigate viral transmission and emerging variants.