The trimethoprim resistance gene dfrK was found to be part of the novel Tn554-related transposon Tn559 integrated in the chromosomal radC gene of a porcine methicillin-susceptible Staphylococcus aureus ST398 strain. While Tn559 and Tn554 had similar arrangements of the transposase genes tnpA, tnpB, and tnpC, the Tn554-associated resistance genes erm(A) and spc were replaced by dfrK in Tn559. Circular forms of Tn559 were detected and suggest the functional activity of this transposon.