A novel trimethoprim resistance gene, designated dfrK, was detected in close proximity to the tetracycline resistance gene tet(L) on the ca. 40-kb plasmid pKKS2187 in a porcine methicillin (meticillin)-resistant Staphylococcus aureus isolate of sequence type 398. The dfrK gene encodes a 163-amino-acid dihydrofolate reductase that differs from all so-far-known dihydrofolate reductases.