The pandemic spread of SARS-CoV-2 and the resulting global healthcare emergency warrants a better understanding of its biology.The potential of SARS-CoV-2 evolution to create novel dangerous variants remain underexplored. Thus, we passaged SARS-CoV-2 in defined conditions and determined its genomic adaptation dynamics. We demonstrate the presence of remarkably stable SARS-CoV-2 quasispecies. We further show that the quasispecies nature of the virus population ensured rapid adaptation of the spike PRRARS motif upon passaging in Vero cells. On the other hand, SARS-CoV-2 replication in HGNC expressing cells led to a reverse mutation at the same site. We observed the emergence of novel mutations in envelope protein PROTEIN upon virus culture in Calu-3 and Caco-2 cells. Finally, we show that the heparan sulfate-binding motif (PRRARS) of the SARS-CoV-2 S protein PROTEIN acted as a determinant of negative growth selection. Overall, our research has far-reaching implications for development of antiviral strategies, suggesting viral quasispecies may facilitate rapid emergence of escape mutants under selection pressure, such as the treatment with antivirals against SARS-CoV-2.