Aminoglycoside resistance is common among strains of Acinetobacter baumannii responsible for nosocomial infections, and inactivation of these antibiotics by enzymatic modification is the main mechanism. Different types of aminoglycoside acetyltransferases (AAC), nucleotidyltransferases (ANT), and phosphotransferases (APH) are synthesized by clinical isolates, and several enzymes can be produced by a single strain. Using a multiplex PCR procedure carried out on bacterial thermolysates, we analyzed the aminoglycoside resistance gene content of strains belonging to eight clusters identified by pulsed-field gel electrophoresis. In a single reaction were combined three primer pairs in order to amplify the genes coding for AAC(6')-Ih, AAC(3)-I, and AAC(3)-II, three primer pairs for the genes coding for ANT(2'')-I, APH(3')-VI, and rRNA 16S as internal control, and finally two primer pairs for the genes coding for AAC(6')-Ib and APH(3')-I. According to the aminoglycoside resistance gene patterns, the strains of the eight clusters were distributed into seven classes. This simple and rapid (< 8 h) fingerprinting technique could be a useful tool for the epidemiological investigation of A. baumannii nosocomial infections.