Purpose: In light of previous studies eliminating the involvement of gene-mediated mechanisms in developing tedizolid resistance, our study elucidates the ability of mutation-mediated mechanisms to confer oxazolidinones cross-resistance in methicillin-resistant Staphylococcus aureus (MRSA). With further investigation of the identified mutations and their relation to tedizolid resistance. Additionally, the involvement of rpoB mutations in acquiring resistance to tedizolid was also investigated. Methods: Five cfr-negative, methicillin-resistant Staphylococcus aureus clinical isolates were subjected to in vitro selection to develop linezolid-resistant mutants. The resultant mutants were tested for acquiring tedizolid cross-resistance, whole genome sequencing was performed twice, followed by variant calling and annotation. Detected mutations were analyzed for their relatedness to the developed resistance. Results: Mutations considered relevant to tedizolid resistance were detected in rpoB gene encoding β-subunit of the RNA polymerase enzyme and rplC gene encoding the 50S ribosomal protein L3. Additionally, mutations in mepB gene, part of the mepRAB operon were detected and believed to contribute to acquiring linezolid resistance. Conclusion: To the best of our knowledge, our findings are the first to report the 50S ribosomal protein L3 mutation Gly152Asp to solely confer cross-resistance to both linezolid and tedizolid oxazolidinones. In addition, we report the emergence of cross-resistance between oxazolidinone antibiotics and rifampin through a single amino-acid substitution occurring within the Rifampin Resistance Determining Region (RRDR). Furthermore, mepB mutations reported in our results support a theory implying a second MepR-independent mechanism regulating the mepRAB operon, and are believed to be responsible for the acquired linezolid resistance in our study.