Acinetobacter baumannii is a species of non fermentative Gram-negative bacteria commonly found in water and soil. This organism was susceptible to most antibiotics in the 1970s. It has now become a major cause of hospital-acquired infections worldwide due to its remarkable propensity to rapidly acquire resistance determinants to a wide range of antibacterial agents. Herein we have determined the mutation frequency of two hot spot residue 83 in gyrA of gyrase and residue 80 in parC of topoisomerase IV and performed a comparative screen the drug resistance ability in 128 clinical isolates of Acinetobacter baumannii in Taiwan region. Low frequency of mutation was found (11.7, 11.7, and 10.2 in gyrA, parC, or both, respectively). Mutation of these sites was not correlated with drug resistance. Our study suggested that mutation of gyrA and parC may play a minor role in quinolone resistance and other mechanisms may contribute to the drug resistance of A. baumannii.