Background ESBL-producing Escherichia coli (ESBL- Ec) is considered a key indicator for antimicrobial resistance (AMR) epidemiological surveillance in animal, human and environment compartments. There is likelihood of ESBL- Ec animal–human transmission but proof of cross-compartment transmission is still unclear. Objectives To characterize ESBL- Ec genetic similarity in various compartments (humans, animals and environment) from a rural area of Madagascar. Methods We collected ESBL- Ec isolates prospectively from humans, animals and the environment (water) between April and October 2018. These isolates were subject to WGS and analysed with cutting-edge phylogenomic methods to characterize population genetic structure and infer putative transmission events among compartments. Results Of the 1454 samples collected, 512 tested positive for ESBL- Ec. We successfully sequenced 510 samples, and a phylogenomic tree based on 179 365 SNPs was produced. Phylogenetic distances between and amongst compartments were indistinguishable, and 104 clusters of recent transmission events between compartments were highlighted. Amongst a large diversity of ESBL- E c genotypes, no lineage host specificity was observed, indicating the regular occurrence of ESBL- Ec transfer among compartments in rural Madagascar. Conclusions Our findings stress the importance of using a phylogenomic approach on ESBL- Ec samples in various putative compartments to obtain a clear baseline of AMR transmissions in rural settings, where one wants to identify risk factors associated with transmission or to measure the effect of 'One Health' interventions in low- and middle-income countries. [ABSTRACT FROM AUTHOR]