Identification of rodents is very difficult mainly due to high similarities in morphology and controversial taxonomy. In this study, mitochondrial cytochrome oxidase subunit I (COI) was used as DNA barcode to identify the Murinae and Arvicolinae species distributed in China and to facilitate the systematics studies of Rodentia. In total, 242 sequences (31 species, 11 genera) from Murinae and 130 sequences (23 species, 6 genera) from Arvicolinae were investigated, of which 90 individuals were novel. Genetic distance, threshold method, tree-based method, online BLAST and BLOG were employed to analyse the data sets. There was no obvious barcode gap. The average K2P distance within species and genera was 2.10% and 12.61% in Murinae, and 2.86% and 11.80% in Arvicolinae, respectively. The optimal threshold was 5.62% for Murinae and 3.34% for Arvicolinae. All phylogenetic trees exhibited similar topology and could distinguish 90.32% of surveyed species in Murinae and 82.60% in Arvicolinae with high support values. BLAST analyses yielded similar results with identification success rates of 92.15% and 93.85% for Murinae and Arvicolinae, respectively. BLOG successfully authenticated 100% of detected species except Leopoldamys edwardsi based on the latest taxonomic revision. Our results support the species status of recently recognized Micromys erythrotis, Eothenomys tarquinius and E. hintoni and confirm the important roles of comprehensive taxonomy and accurate morphological identification in DNA barcoding studies. We believe that, when proper analytic methods are applied or combined, DNA barcoding could serve as an accurate and effective species identification approach for Murinae and Arvicolinae based on a proper taxonomic framework.