Biological evidence is often found at crime scenes that are from human and various unknown organisms. In some cases, species identification is important for an investigation. Therefore, a direct sequencing method that targets three regions of the 16s rRNA gene was examined to classify 14 mammalian samples. For each mammalian DNA sample, PCR products were analyzed by gel electrophoresis with modified universal primers. Direct sequencing was performed using a BigDye Terminator v1.1 Cycle Sequencing kit and an ABI 3130xl Genetic Analyzer. Each sequence was evaluated by a nucleotide BLAST homology search. The top hits for sequence homology for each sample matched the actual species, or closely-related species. In conclusion, this effective method may be used in routine forensic practices to identify the species of unknown mammalian biological samples, such as from blood, body fluid, tissue, hair, and bone.