ABSTRACTLinezolid is often the drug of last resort for serious methicillin-resistantStaphylococcus aureus(MRSA) infections. Linezolid resistance is mediated by mutations in 23S rRNA and genes for ribosomal proteins;cfr, encoding phenicol, lincosamide, oxazolidinone, pleuromutilin, and streptogramin A (PhLOPSA) resistance; its homologuecfr(B); oroptrA, conferring oxazolidinone and phenicol resistance. Linezolid resistance is rare inS. aureus, andcfris even rarer. This study investigated the clonality and linezolid resistance mechanisms of two MRSA isolates from patients in separate Irish hospitals. Isolates were subjected tocfrPCR, PhLOPSAsusceptibility testing, 23S rRNA PCR and sequencing, DNA microarray profiling,spatyping, pulsed-field gel electrophoresis (PFGE), plasmid curing, and conjugative transfer. Whole-genome sequencing was used for single-nucleotide variant (SNV) analysis, multilocus sequence typing, L protein mutation identification,cfrplasmid sequence analysis, andoptrAandcfr(B) detection. Isolates M12/0145 and M13/0401 exhibited linezolid MICs of 64 and 16 mg/liter, respectively, and harbored identical 23S rRNA and L22 mutations, but M12/0145 exhibited the mutation in 2/6 23S rRNA alleles, compared to 1/5 in M13/0401. Both isolates were sequence type 22 MRSA staphylococcal cassette chromosomemectype IV (ST22-MRSA-IV)/spatype t032 isolates, harboredcfr, exhibited the PhLOPSAphenotype, and lackedoptrAandcfr(B). They differed by five PFGE bands and 603 SNVs. Isolate M12/0145 harboredcfrandfexAon a 41-kb conjugative pSCFS3-type plasmid, whereas M13/0401 harboredcfrandlsa(B) on a novel 27-kb plasmid. This is the first report ofcfrin the pandemic ST22-MRSA-IV clone. Differentcfrplasmids and mutations associated with linezolid resistance in genotypically distinct ST22-MRSA-IV isolates highlight that prudent management of linezolid use is essential.