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We determined the prevalence and molecular characteristics of <i>bla</i>_CTX-M-55-positive <i>Escherichia coli</i> (<i>E. coli</i>) isolated from duck–fish polyculture farms in Guangzhou, China. A total of 914 <i>E. coli</i> strains 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%) <i>bla</i>_CTX-M-1G-producing strains were <i>bla</i>_CTX-M-55-positive. MIC results showed that the 177 <i>bla</i>_CTX-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 the <i>bla</i>_CTX-M-55 gene among the <i>E. coli</i>. <i>bla</i>_CTX-M-55 was located on different F18:A-:B1 plasmids with sizes between ~76 and ~173 kb. In addition, the presence of <i>bla</i>_CTX-M-55 with other resistance genes (e.g., <i>tetA</i>, <i>floR</i>, <i>fosA3</i>, <i>bla</i>_TEM, <i>aadA5 CmlA</i> and <i>InuF</i>) on the same F18:A-:B1 plasmid may result in co-selection of resistance determinants and accelerate the dissemination of <i>bla</i>_CTX-M-55 in <i>E. coli</i>. In summary, the F18:A-:B1 plasmid may play an important role in the transmission of <i>bla</i>_CTX-M-55 in <i>E. coli</i>, and the continuous monitoring of the prevalence and transmission mechanism of <i>bla</i>_CTX-M-55 in duck–fish polyculture farms remains important.