Carbapenems are considered last-line agents for the treatment of serious infections caused by Klebsiella pneumoniae, and this microorganism may exhibit resistance to β-lactam antibiotics due to different mechanisms of resistance. We evaluated 27 isolates of K. pneumoniae resistant to carbapenems recovered from inpatients at the University Hospital of Santa Maria-RS from July 2013 to August 2014. We carried out antimicrobial susceptibility, carbapenemase detection, testing for the presence of efflux pump by broth microdilution and loss of porin by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Genetic similarity was evaluated by ERIC-PCR. High levels of resistance were verified by the minimum inhibitory concentration for the antimicrobials tested. The blaKPC gene was present in 89% of the clinical isolates. Blue-Carba and combined disk with AFB tests showed 100% concordance, while the combined disk test with EDTA showed a high number of false-positives (48%) compared with the gold-standard genotypic test. Four isolates showed a phenotypic resistance profile consistent with the overexpression of the efflux pump, and all clinical isolates had lost one or both porins. The ERIC-PCR dendrogram demonstrated the presence of nine clusters. The main mechanism of resistance to carbapenems found in the assessed isolates was the presence of the blaKPC gene.