To obtain the strains resistant to tachyplesin-I, we explored whether tachyplesin-I could induce resistance in three strains by three different methods, which may provide certain theoretical support for research of antibacterial peptides resistant mechanism. Escherichia coli ATCC25922, JM109 and Pseudomonas aeruginosa CCTCC2620 were induced by continuous passages in sub-inhibitory concentration of drugs, microwave and UV mutagenesis techniques. The resistance to tachyplesin-I strains were selected through gradient concentration tachyplesin method, contrasted with the corresponding non-mutagenesis strains, and subsequently, the methods, of which was more available to obtain resistant strains to tachyplesin-I were compared. The experimental results were as follows: (1) No resistance strains to tachyplesin I in three strains were obtained for continuous forty-two generations in sub-inhibitory concentration of tachyplesin-I. (2) The results of microwave and UV mutagenesis indicated that Escherichia coli ATCC25922 was more sensitive to UV than microwave mutagenesis. One mutant resistant to tachyplesin I in Escherichia coli ATCC25922 and nine mutants in Pseudomonas aeruginosa CCTCC2620 were obtained by UV mutagenesis. The MIC values of tachyplesin I for induction strains were four times higher than those of the original strains. But tachyplesin I did not induce resistance in JM109 by UV mutagenesis. Microwave mutagenesis failed to obtain resistance strains in Escherichia coli ATCC25922 and JM109. The above studies have shown that under this research condition, the method of UV mutagenesis is easier to screen the drug-resistant strains. Resistance mechanism of tachyplesin-I-resistant strain induced by mutagenesis needs further research.