Cefoxitin-resistant Escherichia coli and Klebsiella pneumoniae are relatively prevalent in Korea, suggesting dissemination of plasmid-mediated AmpC β-lactamases. In this study, 238 isolates of cefositin-resistant E. coli and K, pneumoniae (not including subspecies ozaenae and rhinoscleromatis) were collected in 2003 from 16 Korean hospitals. The prevalence of plasmid-mediated AmpC β-lactamases was determined by PCR. The AmpC gene alleles detected in E. coli and K. pneumoniae were blaDHA-1, 10 (8.6%) and 93 (76.2%); blaCMY-1-like, 14 (12.1%) and 2 (1.6%); and blaCMY-2-like, 38 (32.7%) and 1 (0.8%) isolates, respectively. The genes identified were blaDHA-1, blaCMY10-like, and blaCMY-2-like, and a new variant, blaCMY-18. Plasmid-mediated AmpC gene allele-positive isolates were present both in large city and in small province hospitals, as well as in isolates from outpatients. The proportions of plasmid-mediated AmpC gene-positive isolates were similar in both expanded spectrum β-lactamase (ESBL)-producing and -nonproducing isolates. In conclusion, DHA-1, CMY-2-like, and CMY-10-like plasmid-mediated AmpC β-lactamase-producing K. pneumoniae and E. coli isolates are widely disseminated in both large city and small province hospitals. Absence of blaCMY-1 and detection of a novel variant of blaCMY-2, blaCMY-18, indicate continued evolution of the prototype genes. Similar proportions of plasmid-mediated AmpC gene-positive isolates in both ESBL-producing and -nonproducing isolates suggest unhindered future spread of these resistances.