Acinetobacter spp. are opportunistic bacterial pathogens primarily associated with hospital-acquired infections and the spread of multidrug resistant Acinetobacter strains is a growing problem in terms of infection control. The aim of this study was to determine the clonal relationship between strains of nosocomial Acinetobacter baumannii by using rep-PCR method. A total of 75 Acinetobacter strains isolated from various clinical samples of the hospitalized patients between October 2011-May 2012 were included in the study. Antibiotic susceptibilities of Acinetobacter isolates were investigated by Kirby-Bauer disk diffusion method according to CLSI guidelines. According to disk diffusion test, the resistance rates for piperacillin, piperacillin-tazobactam, cefepime, ceftazidime, imipenem, meropenem, gentamicin, amikacin, tetracycline, levofloxacin, ciprofloxacin and trimetoprim-sulfamethoxazole were 96%, 96%, 97.3%, 89.3%, 96%, 94.6%, 66.7%, 85.3%, 68%, 82.7%, 97.3% and 89.3% respectively. In this study, 73 (97%) strains were found resistant to three or more than three antibiotics (multidrug resistant). Rep-PCR analysis have shown the presence of eight clones, including two major clones [A (7subtypes), B (3 subtypes)] and six unique clones (C-H). Clone A was found to be the dominant type. Fifty-four (72%) of the 75 Acinetobacter strains belonged to clone A, 13 (17.3%) to clone B, two strains to clone C, D, and one of each to the other clones (E, F, G, H). Clone A was isolated from 71% (20/28), 70% (7/10) and 100% (6/6) of the samples sent from reanimation intensive care unit, surgery ward and internal diseases intensive care units, respectively. The time interval between the first and last strain was eight months. The results of this study indicated an increase in the resistance rates of Acinetobacter strains in our hospital and this increase was attributed to the clonal dissemination of the strains. Strains of the clone A were found to be dominant at the intensive care and other clinics of our hospital. It is contemplated that Acinetobacter strains were scattered as a result of cross transmission and patient transfer among clinics. The rep-PCR method which was used in this study was evaluated as a rapid, easily applicable and successful procedure for epidemiological studies. Clonal distribution of resistant strains in the hospital environment emphasizes the significance of infection control measures.