Background Acinetobacter species infections are increasingly found to cause nosocomial infections, particularly in intensive care units. These pathogens are difficult to eliminate from the hospital environment, and the emergence of multiple-drug-resistant strains complicates patient treatment. In this retrospective study, several strains were analyzed to study the possible spread of pan-resistant strains. Methods Macrorestriction analysis was performed on isolates collected in July 2001 from Johannesburg Hospital and strains collected from a number of hospitals in Johannesburg a year later. Results A strain endemic to Johannesburg Hospital that was cefepime and ceftazidime sensitive in 2001 developed resistance to these antibiotics within 1 year. This and other resistant strains were found to have spread among academic and private hospitals in the area by July 2002. Conclusions The development of resistance is believed to be a response to antibiotic pressure and the spread of resistant strains a result of health care worker and/or patient transfer among hospitals. This snapshot epidemiologic study highlights the need to institute stricter infection control measures to limit the spread of organisms such as Acinetobacter among hospitals.