The prevalence and co-transfer of resistance genes and virulence genes among Escherichia coli producing CMY-2 from feces and flies among twenty large-scale poultry farms in China were investigated. Polymerase chain reaction (PCR) was used to examine for the presence of E.coli producing CMY-2. The genetic relationship between isolates carrying blaCMY-2 gene was determined by pulsed-field gel electrophoresis (PFGE). Antimicrobial susceptibilities to 13 antimicrobials agents of isolates producing CMY-2 were tested by diffusion methods. Characterization of isolates producing CMY-2, including other resistance genes, and virulence genes was detected by PCR. The transmission of resistance and virulence genes was demonstrated by conjugation experiment, PCR-based replicon typing (PBRT). The results showed that 33 isolates were confirmed to carry blaCMY-2 gene with a positive rate of 6.0%, which showed resistance to multiple antimicrobial agents. Fifteen resistance genes conferring resistance to other antimicrobial agents were identified, including blaTEM-1, blaCTX-M-55, blaOXA-1, qnrBDS, aac(6’)-Ib-cr, oqxA, tetA, tetC, sul1, sul2, sul3, rmtB, and flor. Furthermore, four virulence genes including iutA, traT, VagC, and fyuA gene were also detected. Twenty-one transconjuncgants were detected in 33 E.coli Producing CMY-2. The co-transfer of between resistance genes and virulence genes was observed on transferred plasmids of lncA/C or lncI1. This study confirmed the prevalence and co-transfer of resistance genes and virulence genes in isolates of E.coli producing CMY-2, providing a reference for disease prevention and transmission in poultry farms.