Abstract Nirmatrelvir was the first protease inhibitor (PI) specifically developed against the SARS-CoV-2 main protease (3CL pro /M pro ) and licensed for clinical use. As SARS-CoV-2 continues to spread, variants resistant to nirmatrelvir and other currently available treatments are likely to arise. This study aimed to identify and characterize mutations that confer resistance to nirmatrelvir. To safely generate M pro resistance mutations, we passaged a previously developed, chimeric vesicular stomatitis virus (VSV-M pro ) with increasing, yet suboptimal concentrations of nirmatrelvir. Using Wuhan-1 and Omicron M pro variants, we selected a large set of mutants. Some mutations are frequently present in GISAID, suggesting their relevance in SARS-CoV-2. The resistance phenotype of a subset of mutations was characterized against clinically available PIs (nirmatrelvir and ensitrelvir) with cell-based and biochemical assays. Moreover, we showed the putative molecular mechanism of resistance based on in silico molecular modelling. These findings have implications on the development of future generation M pro inhibitors, will help to understand SARS-CoV-2 protease-inhibitor-resistance mechanisms and show the relevance of specific mutations in the clinic, thereby informing treatment decisions. Teaser Understanding how SARS-CoV-2 could counter the antiviral drug nirmatrelvir and what it means for the future of COVID-19 treatment.