Ten bla(KPC-2)-harboring Pseudomonas aeruginosa isolates from hospitals located in five different Colombian cities have been characterized. Isolates were multidrug resistant, belonged to five different pulsotypes, and possessed naturally chromosome-encoded bla(AmpC) and bla(OXA-50) genes and the acquired bla(KPC-2) gene. In most cases, the bla(KPC-2) genes were carried by plasmids of different sizes and were associated with Tn4401b or a new structure containing only part of the Tn4401 sequence. This study revealed that several clones of P. aeruginosa producing bla(KPC-2) are disseminating in Colombia.