Multi-drug resistant Klebsiella pneumoniae isolates are associated with nosocomial infections, in which colonized patients act as a reservoir and source of cross-infection for other patients. In this study, the antimicrobial susceptibility of K. pneumoniae was tested by microdilution using the commercial method MicroScan (Siemens). The genetic relatedness of K. pneumoniae strains was determined by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). PCR experiments were carried out to obtain primer sets and positive PCR products were purified and sequenced. From May 2007 until December 2009, 98 clonally related K. pneumoniae isolates were detected from clinical samples of 38 patients admitted to the University Hospital of Bellvitge, Barcelona, Spain, including 27 admitted to the intensive care unit (ICU). The most important sources of the isolates were: lower respiratory tract (n = 12), urine (n = 12), and blood (n = 11). The strains were resistant to amoxicillin/clavulanic acid, piperacillin/tazobactam, tobramycin, amikacin, and ciprofloxacin, and had diminished susceptibility to cefepime. All the isolates shared a common PFGE pattern related to sequence type 14 after MLST analysis. In K. pneumoniae isolates and their transconjugants, the bla(OXA-1) gene was located in the variable region of a class I integron that also contains the aac(6')Ib-cr gene. Sequencing of the quinolone resistance determinant regions of gyrA and parC revealed a S83F change in GyrA and no changes in ParC.