Ralstonia mannitolilytica strains isolated between February 2002 and March 2004 from 30 episodes of infection in 26 patients at Vienna University Hospital were characterized. Twenty-four of the episodes occurred within a 7 month period, suggesting they were outbreak-related, although no common source of infection was identified. The isolates were assayed using PCR to confirm species identification. Random amplification of polymorphic DNA (RAPD) typing classified the R. mannitolilytica isolates into four distinct genotypes: A/I, B/II, C/III and D/IV (15, 13, 1 and 1 isolates, respectively). API 20NE, VITEK Gram-negative Identification Card plus (GNI+) and VITEK Gram Negative Bacillus Identification (GNB) yielded negative or no acceptable biochemical profile for 4, 11 and 11 isolates, respectively. None of the isolates acidified D-arabitol or mannitol. Two isolates (7 %) were positive for nitrate reduction. All 30 R. mannitolilytica isolates were resistant to desferrioxamine, and 29 were able to grow on BCSA. The most active compounds in vitro were ciprofloxacin and cefepime, whilst only the genotype D/IV isolate was sensitive to gentamicin and amikacin (the remaining 29 isolates being resistant to both).