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Background Acinetobacter baumannii has become one of the most serious causative agents of nosocomial infections due to its significant ability to survive on hospital surfaces. It is mainly an emerging opportunistic pathogen infecting patients in intensive care units. This study was aimed to identify the clinical isolates of A. baumannii and to investigate their heterogeneity using polymerase chain reaction (PCR)-based typing methods. Methods A total of 197 nonduplicate isolates recovered from a wide range of clinical samples were subjected to conventional cultural and biochemical tests. For those isolates that were preliminary identified as A. baumannii, rpoB -based PCR with subsequent restriction fragment length polymorphism (RFLP) using two restriction enzymes ( Tag I and Hae III) was performed to investigate the genetic diversity of the strains and their presumptive relationships with different clinical presentation of the disease caused by this pathogen. Results In total, 50 isolates (25.4%) were identified as A. baumannii using conventional phenotypic methods with subsequent confirmation by rpoB sequencing. RFLP analysis demonstrated five different restriction enzyme patterns, designated as A–E clusters. Most A. baumannii isolates were categorized under Cluster A (32%). We found no significant relationship between clinical presentation and the clustering of the isolates. Conclusion This study showed that the rpoB region possesses high discriminatory power to identify the isolates to the species level. This marker showed high interspecies variability that might be useful for strain typing. The results also suggest the possibility of the existence of a predominant clone of A. baumannii among infected patients in Iran.