Avian pathogenic Escherichia coli (APEC) causes severe economic losses in duck farms through complex, frequent infections and secondary infections. In this study, 73 suspected E. coli strains were isolated from diseased ducks of three provinces in China during 2019-2020, and identified using polymerase chain reaction. The O serotypes, drug susceptibility and molecular subtyping of the strains were also analyzed. The results showed that 9 of the 73 strains belong to classical APEC serotypes, including 1 serotype O1 strain, 2 serotype O2 strains, 2 serotype O18 strains, and 4 serotype O78 strains. Antibiotic susceptibility testing indicated that 69/73 (94.52 %) strains were sensitive to polymyxin B, but less than 6/73 (8.22 %) strains were sensitive to clindamycin, kanamycin, amoxicillin and tetracycline. All strains were multidrug-resistant. Pulsed field gel electrophoresis clustering analysis revealed that these strains shared over 50 % similarity of the band patterns, with seven groups showing 100 % similarity among themselves. Notably, strains from Jiangsu and Jiangxi provinces in 2019 had 100 % similarity in certain groups, with some strains showing identical resistance profiles. Additionally, isolates from Anhui and Jiangsu provinces across different years also exhibited 100 % similarity, suggesting potential crossprovince transmission. In summary, the 73 duck-derived E. coli isolates exhibited strong antibiotic resistance and a broad resistance profile, with genotypes showing polymorphic distribution and epidemiological relevance. The 100 % similarity among some strains within and between provinces underscores the need for epidemiological investigation to inform effective prevention and control measures.