Abstract Objectives Escherichia coli producing metallo-β-lactamases with penicillin-binding protein 3 (PBP3) insertions have reduced susceptibility to aztreonam–avibactam and cefiderocol. Here, we analysed high-quality E. coli genomes for PBP3 insertions. Methods E. coli genomes (n = 167 518) were retrieved from EnteroBase with CheckM2 for quality control, fastANI for species confirmation, multi-locus sequencing typing for sequence type (ST) determination and AMRFinderPlus for resistance gene identification. For PBP3 insertion analysis, we used Prokka for predicting coding sequences, BLAST+ for comparing resulted protein sequences and SnpEff for annotating variants. Results Among the included 159 341 genomes, PBP3 insertions with 11 variants were found in 2.01% (n = 3198). The predominant variant is a duplication of 334–337 amino acids (aa) (94.75%, n = 3030), comprising YRIN (65.92%, n = 2108) and its single-aa-variant YRIK (28.83%, n = 922), followed by a similar PYRI duplication of 333–336 (4.16%, n = 133). The less common ones are a TIPY duplication of 331–334 (n = 24) and its single-aa-variant TVPY’s duplication: TVVPY (n = 1), TVPYTVPY (n = 1) and TVPYPVPY (n = 1). Rare duplications include VGDR of 106–109 (n = 3), ANALNIPL of 114–121 (n = 3), AL of 567–568 (n = 1) and TG of 584–585 (n = 1). Insertion variants were detected across 62 countries on six continents, primarily in human samples, and associated with 85 STs, concentrated in high-risk clones ST410 (29.18%, n = 1923), ST167 (23.40%, n = 1740) and ST405 (10.56%, n = 1334), with 83.32% (n = 2218) encoding metallo-β-lactamase NDM. Conclusions Global spread of E. coli harbouring PBP3 insertion, often with NDM β-lactamase, high-risk ST410, ST167 and ST405 clones and various hosts, underscores the escalating antimicrobial resistance crisis and the urgency for a ‘One Health’ strategy.