Rapid spread of CTX-M type extended-spectrum beta-lactamases (ESBL) between the members of Enterobacteriaceae receives attention increasingly all throughout the world. The aim of this study was to investigate the presence of CTX-M type ESBL in Klebsiella pneumoniae clinical strains by phenotypic and molecular methods. A total of 217 non-repetitive K. pneumoniae strains isolated from clinical specimens (152 urine, 20 sputum, 17 wound swabs, 13 blood, 9 tracheal aspirate, 3 CSF and 3 conjunctival swab samples) of inpatients (n = 128) and outpatients (n = 89) admitted to Pamukkale University Medical Faculty Hospital, between January 2006-January 2007, were included to the study. In vitro antimicrobial susceptibilities were determined by disk diffusion technique in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines, and CT (cefotaxime)/CTL (cefotaxime-clavulanate) containing Etest strips (AB Biodisk, Sweden) were applied for phenotypic detection of cefotaximase production. PCR was performed for the detection of CTX-M genes and the subgroups, while the clonal relatedness of the CTX-M positive isolates was investigated by random amplified polymorphic DNA (RAPD) analysis. While imipenem resistance was not detected in any of the isolates, highest rates of resistance were detected for ampicillin (94%) and cephalothin (64.5%) in 217 K. pneumoniae strains. Using the E-test 39.6% (86/217) of the isolates were found positive, and CTX-M positivity was significantly higher in the strains isolated from inpatients (87.4%) than outpatients (12.6%) (p < 0.001). CTX-M gene was identified in 22.1% (19/86) of the E-test positive isolates. All of the CTX-M positive isolates were identified as CTX-M group-1. The highest resistance rates of CTX-M-1 strains were detected for amoxycillin-clavulanate (94.7%) and netilmicin (89.5%), while the lowest rates were detected for ciprofloxacin (26.3%), trimethoprim/sulphamethoxazole (26.3%) and amikacin (42.1%). RAPD identified 11 different banding patterns among the 19 CTX-M-1 positive isolates, the most frequent clusters being Kp3 (n = 3), Kp4 (n = 3) and Kp5 (n = 3). Five of the 8 isolates from pediatric intensive care unit and 4 of the 5 isolates from other pediatric wards exhibited the same band pattern indicating a possible clonal dissemination. Continuous surveillance of beta-lactamases and the identification of their types in gram-negative enteric bacteria has important clinical impact, since it can often provide valuable information for effective infection control measures and for the choice of appropriate antimicrobial therapy.