The molecular basis and evolution of multidrug resistance were established for 54 isolates of Salmonella enterica serovar Ohio, recorded between 1991 and 2005 in Asturias, a northern region of Spain. All isolates were closely related, as shown by cluster analysis of XbaI-BlnI combined profiles. Of these, 33.3% were resistant to one or more unrelated agent(s). Sulphonamides, streptomycin, tetracycline, and trimethoprim, encoded by sul1, aadA1, tet(A) or tet(B), and dfrA1, respectively, were the most common resistances, but ampicillin (bla(TEM-1)), gentamicin (aacC2 or aacC4), kanamycin (aphA1), and chloramphenicol (catA1) were also detected. Two types of complex genetic elements, carried by large conjugative or mobilizable plasmids, were found in isolates resistant to four or more unrelated agents (multidrug resistant), which accounted for 18.5% of the total: (i) a class 1 integron (1600 bp/dfrA1-aadA1) close to a defective Tn10, both inserted within a Tn21-like element that was carried in some cases by Tn9; (ii) Tn3-bla(TEM-1), which was inserted within a defective Tn1721. These elements have been involved in the development and spread of multidrug resistance in S. enterica Ohio, which was detected between 1994 and 2001. The absence of multidrug resistance in later years could have been connected with the European Union strategies for combating antimicrobial resistance and controlling nontyphoid S. enterica in food-producing animals.