Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae have become more common in the United States and throughout the world. We used pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) to examine the molecular epidemiology of KPC-producing K. pneumoniae isolates sent to the Centers for Disease Control and Prevention (CDC) for reference testing from 1996 to 2008. A dominant strain, sequence type 258 (ST 258), was found and likely accounts for 70% of the CDC's K. pneumoniae PFGE database. Isolates with PFGE patterns related to ST 258 were identified in 10 of the 19 U.S. states currently reporting KPC-producing K. pneumoniae, in addition to one isolate from Israel. KPC subtyping and analysis of the surrounding genetic environment were subsequently performed on 23 representative isolates. Thirteen isolates identified as ST 258 possessed either blaKPC-2 or blaKPC-3 and some variability in the Tn4401 element upstream of the blaKPC gene. Escherichia coli DH10B was successfully transformed by electroporation with KPC-encoding plasmid DNA from 20 of the 23 isolates. Restriction analysis of plasmid DNA prepared from transformants revealed a diversity of band patterns, suggesting the presence of different plasmids harboring the blaKPC gene, even among isolates of the same ST.