In the present study, we describe an outbreak caused by extended-spectrum beta-lactamase (ESBL) producing Klebsiella pneumoniae in the neonatal intensive care unit (NICU) of a tertiary-care hospital. Nosocomial blood-stream infections were detected in three patients hospitalized in NICU. Two of the cases were transferred to NICU due to premature birth and the other due to the presence of cleft palate and retrognathia. K.pneumoniae was isolated on the 5th day of hospitalization of the first patient from umbilical swab and blood culture; on the 15th day of hospitalization of the second patient from blood culture and on the 7th day of hospitalization of the third patient from blood culture. The isolates were identified by automated API Rapid ID 32 Staph (BioMerieux, France) system in addition to conventional laboratory methods. Antibiotic susceptibilities of the isolates were determined by using Kirby-Bauer disk diffusion method according to Clinical and Laboratory Standards Institute (CLSI) criteria. The same antibiotic susceptibility patterns were detected in all isolates. Active surveillance cultures included environmental sampling from surfaces of NICU, laryngoscopes, ventilators and connections of ventilators, stethoscopes, nebulizers, aspiration tubings, disinfectant solutions, couveuse and couveuse distilled water. Two ESBL producing K.pneumoniae strains, presenting the same antibiotic susceptibility pattern with the clinical strains, were isolated from one couveuse distilled water sample and one aspiration tubing. All of the K.pneumoniae isolates were resistant to amoxycillin-clavulonic acid, cefazolin, cefepime, ceftriaxone, ceftazidime, cefuroxime, aztreonam and trimetoprim-sulphametoxazole and susceptible to cefoxitin, imipenem, meropenem, gentamicin, tobramycin, amikacin, netilmisin, tetracycline, ciprofloxacin and chloramphenicol. Arbitrarily primed polymerase chain reaction (AP-PCR) analysis done with M13 primer revealed the same genotype for the patient and environmental K.pneumoniae isolates. It was concluded that AP-PCR which is a simple, rapid and cheap method for the determination of genetic relatedness between isolates, can be applied for the detailed evaluation of nosocomial outbreaks to detect the source of infection and control the dissemination of the outbreak.