Objectives: Klebsiella pneumoniae is an opportunistic pathogen associated with nosocomial infections worldwide. Isolates with a K. pneumoniae carbapenemase (KPC)-producing phenotype show reduced susceptibility to first-choice antibiotics. Between 2012–2013, the largest public tertiary-care hospital in Quito (Ecuador) reported an outbreak of KPC-producing bacteria with more than 800 cases. We developed a molecular epidemiological approach to analyse the clonality of K. pneumoniae isolates recovered from selected hospital services and patient samples. Methods: A retrospective cohort study was performed based on microbial isolates and their corresponding records from the hospital and referred to Instituto Nacional de Investigación en Salud Pública (INSPI). From 800 isolates that were collected between 2012–2013, a total of 100 isolates were randomly selected for this study. Antimicrobial susceptibility testing was performed according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Genotypic detection and phylogenetic relationship analysis were performed by multilocus sequence typing (MLST). The blaKPC carbapenemase gene was also amplified by PCR and was sequenced using Sanger sequencing. Results: Molecular analysis showed that the outbreak had a polyclonal origin with two predominant genotypes, comprising sequence types ST25 and ST258, present in 38 and 36 cases, respectively. These genotypes were found in all studied hospital services including general surgery, intensive care unit and emergency. TheblaKPC-5 gene was the most prevalent blaKPC variant in this study. Conclusion: These data indicate that KPC-producing polyclonal K. pneumoniae are frequent causes of nosocomial hospital outbreaks in South America. Similar genotypes have been reported in Colombia, Argentina, Brazil, North America and Asia.