In this study, two new systems for species identification were developed based on size variation of mitochondrial DNA hypervariable regions among animals: one was a conventional method using non-fluorescent primer sets and agarose gel electrophoresis and the other was an automatic method using fluorescent primer sets and capillary electrophoresis. DNA samples from 18 mammal, four birds, and 19 fish species were amplified using three primer sets specific for mammals, birds, and fishes, respectively. The differences in the sizes of the polymerase chain reaction (PCR) products, ranging from about 350 to 900 bp, permitted us to identify species. These systems were successfully applied to various specimens from several criminal cases. In unknown samples, which were different in size from reference DNA markers, sequencing of the PCR products and subsequent BLAST analysis helped to identify species. Furthermore, the sequence data provided us with information on individuals. Because these species identification methods are very simple, easy, rapid, and exact, they are useful in the field of forensic science.