This study was to characterize plasmid mediated AmpC producing isolates in a tertiary hospital in Osun State.  Ampicillinase class C beta lactamases are class C or group I cephalosporinases that confer resistance to a wide variety of beta-lactam antibiotics including alpha methoxy beta-lactamase (cefoxitin), narrow and broad spectrum cephalosporins, (aztreonam). They are poorly inhibited by beta-lactamase inhibitors like clavulanic acid. All Gram negative isolates were tested for, using cefoxitin disc diffusion method and tested for production of AmpC beta-lactamases phenotypically and genotypically. Plasmid DNA extraction was carried out on the isolates using Kado and Lin method, and genotypic detection of plasmid-mediated AmpC β-lactamases were further carried out using polymerase chain reaction to determine the ampC genes. The results showed that two hundred and four (43.3%) strains were resistant to 3GC, among which 121 (59.3%) were resistant to cefoxitin.  Ninety percent of the isolates were plasmid-mediated. Out of the 204 strains resistant to 3GC, one hundred and eighty nine (40.1%) were AmpC producers. The AmpC producing isolates also concurrently showed multidrug resistance pattern to other antibiotics used. Forty-five percent (45%) isolates were positive for CMY, 38% for FOX while only 31% were amplified for ACC group. This study has shown that the isolates were found to exhibit a high degree of drug resistance and the detection rate of plasmid mediated AmpC producing isolates was high in the study area. Proper antibiotic policy is required to limit the spread of plasmid mediated AmpC producing organism because it can lead to a therapeutic dead in the nearest future.