Mycoplasma gallisepticum (MG) isolations in live chickens have been made from swabs obtained primarily from the trachea or nasal exudates. As tracheal swabs are often contaminated with feed and because tracheal swabbing may be stressful to the bird, this study was conducted to determine if swabs from the choanal cleft (palatine fissure) would yield MG isolation rates comparable to MG isolation rates of swabs taken from the trachea. Commercial Leghorns from 17 to 22 weeks of age were inoculated via eyedrop with the F strain of MG. Swabs were made from the trachea or the choanal cleft region (palatine fissure) when the chickens were 58 to 63 weeks of age; MG was isolated from 15 of 101 tracheal swab samples and from 51 of 108 choanal cleft swab samples. This study indicates that swabs taken from the choanal cleft region yield higher isolation rates and are more easily obtained than tracheal swabs.