Pulsed-field gel electrophoresis typing of 60 extended-spectrum beta-lactamase-producing Klebsiella pneumoniae (ESBLKp) isolates obtained in a neonatal intensive care unit during an outbreak indicated the dissemination of two major bacterial genotypes associated with colonization and invasive disease: one composed by aminoglycoside-resistant isolates and the other by aminoglycoside-susceptible isolates. A urease-negative phenotype was observed among aminoglycoside-resistant ESBLKp. Six pairs of isolates from gastrointestinal (GI) colonization and isolates from invasive disease that occurred 3-23 days later were shown to belong to the same genotype, reinforcing a direct association between colonization and subsequent disease. These data indicate that screening for ESBLKp GI colonization in an outbreak setting may be useful to detect neonates at a higher risk of invasive disease.