Background: Acinetobacter baumannii is responsible for a variety of nosocomial infections, especially in intensive care unit patients. Nosocomial outbreaks due to carbapenem-resistant A. baumannii strains have been reported in many countries, including Greece. The aim of the present study was to determine the trends of molecular epidemiology of carbapenem-resistant A. baumannii isolates in a 750-bed hospital in Thessaloniki, Greece, during 2009. Methods: The study included 39 carbapenem-resistant A. baumannii isolates collected from patients hospitalized in the General Hospital Papageorgiou during 2009. They were tested for the presence of Ambler class D carbapenemases and class 1 integrons, and they were typed by pulsed-field gel electrophoresis. Results: The bla(OXA-58) gene was detected in all A. baumannii isolates. Among the 39 isolates, 18 were carrying a 2.2 kb integron, 18 were carrying a 2.5 kb integron, and 3 isolates had no class 1 integrons. Two different clones, each divided further into two subclones, were observed. Comparing the clones detected in 2009 with those of former years (2001-2008), a significant difference was observed: three clones have disappeared, two clones continued to circulate in the hospital, while a new subclone emerged in February 2009. Conclusions: A change was seen in the molecular epidemiology of carbapenem-resistant A. baumannii isolates during 2009. Molecular epidemiology studies provide useful data for the distribution of resistant bacteria in order to design effective prevention and control measures. Hippokratia 2011; 15 (4): 343-345