The purpose of the present study was to investigate the diversity of the genes encoding aminoglycoside-modifying enzymes and their association with class 1 integrons in three pan-European clones of Acinetobacter baumannii. The study collection included 106 multidrug-resistant strains previously allocated to clone I (n = 56), clone II (n = 36) and clone III (n = 6) and a heterogeneous group of other strains (n = 8), using AFLP fingerprinting and ribotyping. The strains were from hospitals of the Czech Republic (n = 70; collected 1991–2001) and 12 other European countries (n = 36; 1982–1998). Using PCR, at least one of the following aminoglycoside-resistance genes was detected in 101 (95 %) strains: aphA1 (n = 76), aacC1 (n = 68), aadA1 (n = 68), aphA6 (n = 55), aadB (n = 31), aacC2 (n = 7) and aacA4 (n = 3). A combination of two to five different resistance genes was observed in 89 strains (84 %), with a total of 12 different combinations. PCR mapping revealed that aacC1, aadA1 and aacA4 were each associated with a class 1 integron, as was the case with aadB for six strains of clone III. Six different class 1 integron variable regions were detected in 78 strains (74 %), with two predominant regions (2.5 and 3.0 kb) in two sets of 34 strains each. The 3.0 kb region contained five gene cassettes (aacC1, orfX, orfX, orfX′, aadA1) and differed from the 2.5 bp region only by one additional orfX cassette. These two integron regions were confined to clones I and II and were found in strains isolated in seven countries between 1982 and 2001. The clone III strains were homogeneous both in resistance genes and in integron variable regions, whereas clones I and II showed a remarkable intraclonal diversity of these properties, with no clear-cut difference between the two clones. Yet, within the Czech clone I and II strains, the diversity of resistance genes and integron structures was limited as compared to those from other countries. The occurrence of identical resistance genes, gene combinations and class 1 integrons associated with these genes in clonally distinct strains indicates that horizontal gene transfer plays a major role in the dissemination of aminoglycoside resistance in A. baumannii.