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This study characterized the occurrence of carbapenem resistance of Acinetobacter baumannii isolates in a tertiary care hospital in Saudi Arabia. From January 2010 until February 2012, Acinetobacter spp. isolates were collected from different wards and were identified using Vitek 2 system and 16S rRNA gene sequencing. Vitek 2 system and Etest were used for susceptibility testing. PCR and Pulse field gel electrophoresis (PFGE) were used for detecting and typing genes associated with carbapenem resistance. A total of 141 isolates were identified as A. baumannii. A total of 46 (32.6%) isolates were carbapenem-resistant Acinetobacter baumannii (CRAB) isolates and had wild diversity by PFGE. Metallo ?-lactamase confirmatory test was positive for 43 isolates with negative PCR for blaIMP and blaVIM. Among the 46 CRAB strains, 37 isolates harbored blaOXA-23 which was encoded downstream of ISAba1 and 1 isolate had ISAba1 encoded upstream blaOXA-51. These data reveal that the interhospital transmission of CRAB isolates was apparently insignificant. BlaOXA-23 adjacent to ISAba1 was the main mechanism of carbapenem resistance in these isolates. To our knowledge, this is the first molecular study characterizing carbapenem resistance in A. baumannii in the Eastern Province of Saudi Arabia.