Purpose: To characterize clinical isolates of Klebsiella pneumoniae by phenotypic and genotypic methods. Material/methods: Over a 12-month period, 52 isolates of K. pneumoniae were isolated. Of these 52 isolates, 7 were isolated over a period of 21 days from a suspected outbreak in the Neonatal Intensive Care Unit (NICU) and 45 from sporadic cases occurring in different wards of hospital were analysed. Results: The prevalence of K. pneumoniae isolates was 4% (52/1295). Quinolones, aztreonam and amikacin showed the greatest efficacy showing >85% sensitivity. Of the 52 isolates of K. pneumoniae, 8 (15.4%) isolates were positive for ESBL-production. Among the ESBL-producing K. pneumoniae, two out of 8 (25%) and 6 out of 8 (75%) were positive for (bla)(SHV) and (bla)(CTX-M) genes respectively. Ribotyping identified 30 distinct ribogroups among 52 isolates evaluated. Seven NICU outbreak isolates were divided into 2 ribotypes, as many as 6 belonged to one ribotype while one isolate which was isolated a week later was of a different ribotype, indicating the termination of the outbreak in the NICU. The outbreak in the NICU thus, was shown to have been caused by a single clone. Conclusions: A high discriminatory power, ease of interpretation coupled with excellent reproducibility and stability make ribotyping a very useful technique for investigating the molecular epidemiology of nosocomial infections caused by K. pneumoniae. A regular surveillance of hospital associated infections including monitoring antibiotic sensitivity pattern of K. pneumoniae, ESBL-production and molecular characterization is mandatory to control the spread of multidrug-resistant and ESBL-producing K. pneumoniae and for epidemiological purposes especially in outbreak situations. (C) 2014 Published by Elsevier Urban & Partner Sp. z o.o. on behalf of Medical University of Bialystok.