From December 2004 to March 2005, 27 Klebsiella pneumoniae clinical isolates that were positive by the imipenem-EDTA double-disk synergy test and that exhibited a single macro-restriction pattern were recovered in two distinct Greek hospitals. The isolates carried a transferable bla(VIM-1) metallo-beta-lactamase gene in a class 1 integron. Reverse transcriptase PCR showed that the gene was similarly expressed in low- and high-level carbapenem-resistant isolates, indicating the existence of additional resistance mechanisms. The clonal spread of VIM-1-producing K. pneumoniae strains in distinct regions where up to now bla(VIM-2) and bla(VIM-4) alleles were common is worrisome.