A total of 78 gentamicin-resistant Escherichia coli strains from swine (27) and cattle (51) were characterized by phenotypic resistance, presence of selected aminoglycoside resistance genes, class 1 integrons and gene cassettes, and pulsed-field gel electrophoresis. Gentamicin resistance was mainly encoded by the ant(2'')-I gene that was found in 76% of all the strains investigated, whereas the aac(3)-IIa gene was found in 14%. The ant(2'')-I gene was predominant in strains from cattle, whereas the porcine strains contained both ant(2'')-I, aac(3)-IIa, and the aac(3)-IVa genes. The pulsed-field gel electrophoresis (PFGE) investigation indicated a close clonal relationship in half of the bovine strains whereas the remaining E. coli were unrelated. Among the E. coli investigated, 20% contained class 1 integrons. Genes encoding resistance to trimethoprim (dhfrI, dhfrIb, and dhfrVII), gentamicin, tobramycin, and kanamycin (ant(2'')-Ia streptomycin and spectinomycin (ant(3'')-Ia) and streptothricin (sat1) were identified as gene cassettes. The most prevalent gene cassettes were ant(3'')-Ia (11 isolates) and the dhfrI (nine isolates).