Mycoplasma synoviae is an economically significant pathogen of commercial poultry worldwide, which generally produces subclinical to mild upper respiratory tract or synovitis infections with lateral and vertical transmission. The effect of tiamulin hydrogen fumarate treatment on agent persistence in the trachea ofM. synoviae infected layers was evaluated. Two groups of eight, 30 week old M. synoviae infected (PCR positive) cómmercial layers (lsabrown line), were studied. The first group received two three day treatments of tiamulin (33mg/kg administered in the drinking water) with a 27 day interval, while the second group was not medicated. Tracheal swabs were taken weekly for two months. Ali 16 samples were tested by PCR, using pools of four tracheal swabs, and found positive to M. synoviae confirming pathogen persistence in the trachea of both treated and untreated birds.