Objective To compare Chelex 100 resin method (six different processing procedures) with the commercial automated magnetic bead-based DNA purification method (the standard method for DNA extraction) for the extraction of genomic DNA from animal tissues, and to identify a simple and rapid method for on-site testing of genomic DNA from animal tissue samples. Methods An appropriate amount of rodent liver tissue was ground or cut prior to adding 5% Chelex 100 resin suspension. The mixture of homogenized tissue with 5% Chelex 100 resin suspension was treated according to six different processing procedures of lysis and adsorption. After centrifugation or static treatment, the concentration and purity of genomic DNA in supernatant were measured. Using the genomic DNA as the templates, the primers of mitochondrial cytochrome c oxidase subunit I (COI) gene for rodents, the universal primers for Bartonella spp. and the TaqMan probe were used to amplify the COI gene fragment and the tmRNA gene fragment of Bartonella spp.. The concentration and purity of genomic DNA and the quantification cycle (C_q) value were compared between the six processing procedures based on Chelex 100 resin as well as between the Chelex 100 resin method and the magnetic bead method. Results The concentration of the DNA templates obtained using the six different processing procedures (C1-C6) based on Chelex 100 resin ranged from 203.93 ng/μl to 769.86 ng/μl, which was higher than that obtained using the magnetic bead method. The A260/A280 ratios of DNA templates extracted using the Chelex 100 resin method and the magnetic bead method ranged from 1.25 to 1.47 and 1.85 to 1.95, respectively. There was no difference in A260/A280 ratio between the C1-C6 based on Chelex 100 resin, but the purity of DNA templates extracted using the Chelex 100 resin method was lower than that of DNA templates extracted using the magnetic bead method. The electrophoretograms showed that the bands of the DNA templates extracted using the magnetic bead method were more distinct than those extracted using the Chelex 100 resin method and there was a clear and accurate band for amplification product of the COⅠ gene. The quantitative real-time PCR amplification results showed that the DNA templates obtained using the Chelex 100 resin method had a normal C_q value, which was slightly higher than that of the DNA templates obtained using the magnetic bead method. Conclusion The Chelex 100 resin method for extracting genomic DNA of animal tissues is simple and efficient, which is suitable for conventional PCR and TaqMan fluorescent probe-based quantitative real-time PCR assay to identify host animal species and directly detect pathogens in animal tissue samples.