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This study investigated the existence of sulfonamides and colistin resistance genes among extended‐spectrum beta‐lactamase (ESBL)‐producing Escherichia coli recovered from fish gut in Vietnam and evaluated the susceptibility patterns of the ESBL‐producing E. coli to relevant antimicrobials. A total of 88 ESBL‐producing E. coli isolates were analysed for the presence of the ESBLs, sul (1, 2, 3) and mcr (1–3) genes by PCR. Antimicrobial resistance phenotypes of isolates were determined by disc diffusion. Results showed that: (i) A high prevalence of 94·3% of sulfonamide resistance was observed in 88 isolates. Moreover, the existence of 2·3% of ESBL‐producing E. coli harbouring mcr‐1 gene were detected; (ii) The phylogenetic types A and B1 were most frequent, and the blaCTX‐M group1 and blaTEM genes encoding ESBL were detected in 47·7% of the isolates; (iii) ESBL‐producing E. coli harbouring mcr‐1 gene exhibited resistance to 11 antibiotics. The existence of mcr‐1 and sul1,2,3 genes and the extremely high level of multiple drug resistance in all ESBL‐producing E. coli isolates obtained from sampled fish in Vietnam is a major concern. Therefore, it is imperative to monitor ESBL‐producing E. coli in the river waters of Vietnam. Significance and Impact of the Study: Although extended‐spectrum beta‐lactamase (ESBL)‐producing Escherichia coli have been thoroughly investigated in Vietnamese society, distribution of ESBL‐producing E. coli in fisheries has not been. This is the first study reporting ESBL‐producing E. coli isolates harbouring sul and mcr‐1 genes from fish gut in the Mekong delta, Vietnam. The existence of ESBL, sulfonamide and colistin resistance genes and high levels of multiple drug resistance in resistant E. coli isolates obtained from fish gut in this region is a major concern. [ABSTRACT FROM AUTHOR]