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Mitochondrial DNA is an important tool for human identification and is used to differentiate between human and animal blood at the crime scene, because in extreme conditions nuclear DNA is severely destroyed while Mitochondrial DNA contains multiple copies (200–2000) per cell and resists harsh and more stable conditions. Seventy-two blood samples were collected from humans (Homo sapiens), sheep (Ovis aries), goats (Capra hircus), and cows (Bos taurus) (18 blood samples for each). All blood samples were withdrawn by a technician and 5 ml were aspirated using an aseptic technique and transferred to EDTA-Na2 tubes. They were mixed well and stored in a refrigerator. The collection took 2 weeks (May 15, 2019–May 30, 2019). All samples were collected from Al-Diwanyia city. The results of PCR testing revealed that the primer pairs were specific and non-specific products did not appear for all samples. The amplification of Homo sapiens mitochondrial DNA with primer pairs of other (Ovis aries, Capra hircus, and Bos taurus) and amplification of each with primer pairs of another genus gave negative results, and this is primary evidence for primer pair specificity. The amplicon of 16S rRNA gene of Homo sapiens was 1200 bp, Ovis aries was 1060 bp, Capra hircus was 820 bp, and Bos taurus was 1300 bp. The sequencing revealed that no cross-reactivity of designed primer pairs and the PCR assay based on the designed primer pairs will be simple, fast, sensitive, specific, and cost-effective. There is sensitivity, specificity, and accuracy in the designed species-specific primer pairs and applicability of the designed primer pairs in forensics to investigate blood spots or evidence belonging for human, sheep, goat, and cow.