The continuous and rapid development of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus remains a health concern especially with the emergence of numerous variants and mutations worldwide. As with other RNA viruses, SARS-CoV-2 has a genetically high mutation rate. These mutations have an impact on the virus characteristics, including transmissibility, antigenicity and development of drug and vaccine resistance. This work was pursued to identify the differences that exist in the papain-like protease (PL^Pro) from 58 Saudi isolates in comparison to the first reported sequence from Wuhan, China and determine their implications on protein structure and the inhibitor binding. PL^pro is a key protease enzyme for the host cells invasion and viral proteolytic cleavage, hence, it emerges as a valuable antiviral therapeutic target. Two mutations were identified including D108G and A249V and shown to increase the molecular flexibility of PL^Pro protein and alter the protein stability, particularly with D108G mutation. The effect of these mutations on the stability and dynamic behavior of PL^Pro structures as well as their effect on the binding of a known inhibitor; GRL0617 were further investigated by molecular docking and dynamic simulation.