In this study a multilocus sequence typing (MLST) scheme for Acinetobacter baumannii was developed and evaluated by using 40 clinical A. baumannii isolates recovered from outbreaks in Spanish and German hospitals during the years 1990 to 2001, as well as isolates from other European hospitals and two DSMZ reference strains of A. baumannii. For comparison, two isolates of Acinetobacter species 13 (sensu Tjernberg and Ursing), two clinical isolates, and three DSMZ strains of A. calcoaceticus (both belonging to the A. calcoaceticus-A. baumannii complex) were also investigated. Primers were designed for conserved regions of housekeeping genes, and 305- to 513-bp internal fragments of seven such genes—gltA, gyrB, gdhB, recA, cpn60, gpi, and rpoD—were sequenced for all strains. The number of alleles at individual loci ranged from 6 to 12, and a total of 20 allelic profiles or sequence types were distinguished among the investigated A. baumannii strains. The MLST data were in high concordance with the epidemiologic typing results generated by pulsed-field gel electrophoresis and amplified fragment length polymorphism fingerprinting. The MLST scheme provides a high level of resolution and an excellent tool for studying the population structure and long-term epidemiology of A. baumannii.