To further understand the role of low-energy N⁺ implantation in the phylogenetic evolution and characterization of drug resistance in Escherichia coli (E.coli), this study used low-energy N⁺ ion implantation to screen for drug resistant E.coli. The 16S rRNA gene sequences were obtained through de novo genome sequencing, and the drug resistance characteristics of mutant strains were assessed using the K-B method. Twenty-five drug-resistant strains were obtained by mutagenesis. The 16S rRNA in five mutant strains had a point mutation (A257C) or a gene deletion in C1, V1, V2, V6-V9, and C6-C9 regions, respectively. GC content increased and the mutation rate reached 0.4%-0.6%. The results indicate that low-energy N⁺ ion implantation could trigger mutations and drive 16S rRNA gene evolution in E.coli., which could accelerate development of antibiotic resistance in E.coli.