<title>Abstract</title> Escherichia coli and Klebsiella pneumoniae are resistant to third-generation cephalosporins, carbapenems, colistin, and tigecycline, becoming a major public health priority. Mainly within the developing world. However, their genomic epidemiology and possible determinants of resistance remain to be elucidated. Thus, this study aimed to perform genomic characterizations for E. coli and K. pneumoniae that are resistant to last-line antibiotics, isolated from humans, and poultry, ther with a dairy-farm environment within Ecuador. This study analyzed, at the genomic level, nine 3CG-resistant E. coli isolates harboring the mcr-1 gene (six from poultry farms, two from human infections, and one from dairy-farm compost), together with ten isolated colistin and carbapenem-resistant K. pneumoniae clinical samples. E. coli isolates of human origin belonged to ST609 and phylogroup A, while poultry and compost isolates belonged to differing STs and phylogroups A, B1, E, and F. K. pneumoniae isolates were ST13 (five isolates), ST258 (four isolates) and ST86 (one isolate). Within E. coli isolates, blaCTX-M-55, blaCTX-M-65, blaCTX-M-15, and blaCTX-M-2 genes were identified. This study also identified blaCMY-2 and blaKPC-3 (the latter in a carbapenem-susceptible isolate). The plasmid-borne mcr-1.1 gene was identified across all E. coli isolates within an IncI2 plasmid (reference for assembling CP018669). Tigecycline-reduced susceptibility or resistance was related to missense-sense mutations in marA and acrA genes. Within K. pneumoiae, blaCTX-M-15 and blaCTX-M-65 blaKPC-2 and blaKPC-3, encoding ESBLs and carbapenemases, were detected. The blaKPC-2 genes were identified in a ̴ 10-kb Tn4401 transposon, with genetic context being: tnpR-tnpA-istA-istB-blaKPC-2-tnpA. This investigation identified a nonsense mutation in mgrB (K3*) and several mis-sense mutations within arnB, eptA, pmrB, pmrJ, and phoQ, linked to colistin resistance. Tigecycline resistance was linked to nonsense and mis-sense mutations within the ramR gene. Additionally, this study identified several integrons, including Int191(5’CS-dfrA14-3’CS), which was the most prevalent integron among E. coli and K. pneumoniae isolates, followed by Int0 (5’CS-3'CS) and int18 (5’CS-dfrA1-3'CS). These results could contribute to future MDR E. coli and K. pneumoniae epidemiological studies.