Mitochondrial DNA (mtDNA) is a hereditary material located in mitochondria and is normally maternally inherited. Mutational analysis performed on mtDNA proved that the mutations are closely related with a number of genetic illnesses, besides being exploitable for forensic identification. Those findings imply the importance of mtDNA in the scientific field. MtDNA can be found in abundance in tooth dentin where it is kept protected by the enamel, the hardest outer part of the tooth. In this study, two techniques of mtDNA extraction were compared to determine the efficacy between the two techniques. Teeth used for the study was collected from Dental Clinic, Hospital Universiti Sains Malaysia. After the removal of tooth from the tooth socket of the patient, the tooth was kept at -20C until use. Later, pulp tissue and enamel was excised using dental bur and only the root dentin was utilized for the isolation of mtDNA by crushing it mechanically into powdered form. MtDNA was extracted using the two published methods, Pfeifer and Budowle and then subjected to spectrophotometry DNA quantification and purity, Polymerase chain reaction (PCR) amplification of hypervariable-two region of mtDNA, followed by DNA sequencing to analyze the reliability of the extraction techniques. In conclusion, both techniques proved to be efficient and capable for the extraction of mtDNA from tooth dentin.