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This study aimed to investigate and compare the proportion of AMR Escherichia coli (E. coli) between urban (Dompe in the Western province) and rural (Dambana in the Sabaragamuwa province) areas in Sri Lanka. The overall hypothesis of the study is that there is a difference in the proportion of AMR E. coli between the urban and the rural areas. Faecal samples were collected from healthy humans (n = 109), dairy animals (n = 103), poultry (n = 35), wild mammals (n = 81), wild birds (n = 76), soil (n = 80) and water (n = 80) from both areas. A total of 908 E. coli isolates were tested for susceptibility to 12 antimicrobials. Overall, E. coli isolated from urban area was significantly more likely to be resistant than those isolated from rural area. The human domain of the area had a significantly higher prevalence of AMR E. coli, but it was not significantly different in urban (98%) and rural (97%) areas. AMR E. coli isolated from dairy animals, wild animals and water was significantly higher in the urban area compared with the rural area. There was no significant difference in the proportion of multidrug resistance (MDR) E. coli isolated from humans, wild animals and water between the two study sites. Resistant isolates found from water and wild animals suggest contamination of the environment. A multi‐sectorial One Health approach is urgently needed to control the spread of AMR and prevent the occurrences of AMR in Sri Lanka. [ABSTRACT FROM AUTHOR]