A total of 220 gram-negative isolates showing distinct beta-lactam resistance profiles recovered in U.S. medical centers during the MYSTIC Program 2007 were evaluated to determine the presence of selected beta-lactamase genes. CTX-M-encoding genes, considered rare in the United States, were detected in 38.8% (28/70; three species) of the extended spectrum beta-lactamase-positive isolates and were observed in 80.0% of the participating hospitals. CTX-M-14 and -15 were found in multiple institutions (eight and nine medical centers, respectively), and CTX-M-3 was detected in only one isolate. The OXA-2 and -10 were identified in nine Enterobacteriaceae strains, and plasmid-mediated AmpC enzymes CMY-2 and FOX-5 were identified in six and four isolates, respectively, displaying negative clavulanate inhibition. Genes encoding OXA-23 and -24 were detected in 30.0% (15/50) of carbapenem-resistant Acinetobacter spp. strains. Retrospective sampling showed that these OXA enzymes were present since 2004 in the MYSTIC Program isolates. The KPC serine carbapenemases were observed in the majority of the carbapenem-resistant Enterobacteriaceae (usually Klebsiella pneumoniae), confirming an epidemic problem in the New York City area. The association of beta-lactamase production and transferable quinolone resistance genes (qnr; 6.7%) in Enterobacteriaceae strains was higher than previously reported. This study illustrates the emergence and rapid dissemination of some beta-lactamases, such as CTX-M, broad-spectrum oxacillinases, and serine carbapenemases, that compromised the treatment of gram-negative infections in numerous U.S. hospitals participating in the MYSTIC Program in 2007.