In Brazil, Salmonella enterica serovar Infantis resistant to various antimicrobials, including cephalosporins, has been identified as an etiological agent of severe gastroenteritis in hospitalized children since 1994. In this study, 35 serovar Infantis strains, isolated from children admitted to four different Rio de Janeiro, Brazil, hospitals between 1996 and 2001, were characterized by pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility testing in order to determine their genetic relatedness and antimicrobial resistance profiles. Thirty-four serovar Infantis strains were resistant to at least two antibiotic classes, and all 35 strains were susceptible to fluoroquinolones, cephamycin, and carbapenem. Extended-spectrum beta-lactamase (ESBL) screening by double-disk diffusion indicated that 32 serovar Infantis strains (91.4%) produced beta-lactamases that were inhibited by clavulanic acid. Antimicrobial resistance gene profiles were determined by PCR for a subset of 11 multidrug-resistant serovar Infantis strains, and putative ESBLs were detected by isoelectric focusing. Ten serovar Infantis strains carried bla(TEM), catI, ant(3")Ia and/or ant(3")Ib, sulI and/or sulII, and tet(D) genes as well as an integron-associated aac(6')-Iq cassette. Eight strains possessed at least four different beta-lactamases with pI profiles that confirmed the presence of both ESBLs and non-ESBLs. Our PFGE profiles indicated that 33 serovar Infantis strains isolated from Rio de Janeiro hospitals came from the same genetic lineage.