Acinetobacter baumannii is generally the most frequently isolated Acinetobacter species. Sequence analysis techniques allow reliable identification of Acinetobacter isolates at the species level. Forty-eight clinical isolates of Acinetobacter spp. were obtained from blood cultures at Tohoku University Hospital. These isolates were identified at the species level by partial sequencing of the RNA polymerase β-subunit (rpoB), 16S rRNA, and gyrB genes. Then further characterization was done by using the PCR for detection of OXA-type β-lactamase gene clusters, metallo-β-lactamases, and carO genes. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing were also performed. The most frequent isolate was Acinetobacter soli (27.1%). Six of the 13 A. soli isolates were carbapenem nonsusceptible, and all of these isolates produced IMP-1. PFGE revealed that the 13 A. soli isolates were divided into 8 clusters. This study demonstrated that A. soli accounted for a high proportion of Acinetobacter isolates causing bacteremia at a Japanese tertiary hospital. Non-A. baumannii species were identified more frequently than A. baumannii and carbapenem-nonsusceptible isolates were found among the non-A. baumannii strains. These results emphasize the importance of performing epidemiological investigations of Acinetobacter species.