The recent emergence of mobile-tigecycline resistance tet(X) genes in human and animals in China seriously threats the clinical utility of tigecycline. Here we focused on the isolation and molecular characterization of plasmid-mediated tigecycline resistance tet(X4)-positive E. coli from different sources in Pakistan using MinION and Illumina sequencing. The tet(X4) gene was detected in four E. coli isolates from poultry, chicken meat, wild bird and the slaughterhouse wastewater in Pakistan. Co-existence of colistin resistance mcr-1 gene was also detected in three isolates. The four isolates belonged to different sequence types and the tet(X4) gene was located on plasmids ranging from 12,331 bp to 113,610 bp belonging to IncFII and IncQ replicon types with two genetic contexts ISCR2-tet(X4)-abh-ISCR2-lysR-floR-virD2 and ΔISCR2-abh-tet(X4)-ISCR2-virD2-floR, respectively. In all the four E. coli strains, tet(X4) was transferable by conjugation to E. coli J53 host strain. In addition, three of four strains transferred tet(X4) to a wild-type carbapenem resistant E. coli strain. To our knowledge, this is the first report of the emergence of plasmid-mediated tet(X4) gene from Pakistan. The convergence of tigecycline and colistin resistance in South Asia is a serious threat to human health.