We determined the prevalence and molecular characteristics ofblaCTX-M-55-positiveEscherichia coli(E. coli) isolated from duck–fish polyculture farms in Guangzhou, China. A total of 914E. colistrains were isolated from 2008 duck and environmental samples (water, soil and plants) collected from four duck fish polyculture farms between 2017 and 2019. Among them, 196 strains were CTX-M-1G-positive strains by PCR, and 177 (90%)blaCTX-M-1G-producing strains wereblaCTX-M-55-positive. MIC results showed that the 177blaCTX-M-55-positive strains were highly resistant to ciprofloxacin, ceftiofur and florfenicol, with antibiotic resistance rates above 95%. Among the 177 strains, 37 strains carrying the F18:A-:B1 plasmid and 10 strains carrying the F33:A-:B- plasmid were selected for further study. Pulse field gel electrophoresis (PFGE) combined with S1-PFGE, Southern hybridization and whole-genome sequencing (WGS) analysis showed that both horizontal transfer and clonal spread contributed to dissemination of theblaCTX-M-55gene among theE. coli.blaCTX-M-55was located on different F18:A-:B1 plasmids with sizes between ~76 and ~173 kb. In addition, the presence ofblaCTX-M-55with other resistance genes (e.g.,tetA,floR,fosA3,blaTEM,aadA5 CmlAandInuF) on the same F18:A-:B1 plasmid may result in co-selection of resistance determinants and accelerate the dissemination ofblaCTX-M-55inE. coli. In summary, the F18:A-:B1 plasmid may play an important role in the transmission ofblaCTX-M-55inE. coli, and the continuous monitoring of the prevalence and transmission mechanism ofblaCTX-M-55in duck–fish polyculture farms remains important.