Very little is known about the prevalence and composition of various types of extended-spectrum β-lactamases (ESBL) in pediatric patients. The aims of this study were the following: (i) to determine the prevalence of ESBLs among Enterobacteriaceae in a tertiary-care pediatric population; (ii) to characterize the genetic composition of the identified ESBL enzymes; and (iii) to determine the relative prevalence of CTX-M enzymes and Escherichia coli ST131 strains among ESBL-producing isolates in the same pediatric patient population. Among the 1,430 Enterobacteriaceae isolates screened for elevated MICs to cefotaxime and/or ceftazidime from pediatric patients during a 1-year period, 94 isolates possessed at least one ESBL gene. CTX-M was the most commonly isolated ESBL type, consisting of 74% of all ESBLs versus 27% TEM and 24% SHV enzymes. Sequence analysis and probe-specific real-time PCR revealed that the majority (80%) of the CTX-M-type ESBLs were CTX-M-15 enzymes, followed by CTX-M-14 (17%) and CTX-M-27(2.8%). Multilocus sequence typing (MLST) and repetitive PCR analyses revealed that the relative prevalence of ST131 among ESBL-producing E. coli isolates is 10.2%. This study highlights the growing problem of ESBL resistance in pediatric Enterobacteriaceae isolates and demonstrates a transition toward the predominance of CTX-M-type enzymes among ESBL-producing Enterobacteriaceae organisms causing pediatric infections.