Identification of species in animal-derived foods is a crucial component of its management. Legal, financial, religious, and public health considerations all apply to food adulteration, particularly in the case of beef meat. Food adulteration mostly includes replacement of low price ingredient in meat products to get unlawful higher benefits. To protect consumers from meat adulteration, various methods have been investigated. The DNA-based techniques are rapid, greater resistance to degradation, adequate for the detection of small amounts of DNA in complex handled food varieties. This work was designed to investigate different concentrations of beef meat adulterations with other meat types (equine, sheep, dog, and pork) using multiplex PCR. Fresh meat samples of different animal species cattle, donkey, pork and dog were collected for detection of their adulteration. Meat species samples were minced to make meat mixture for mimicking adulteration. The multiplex PCR assay for five meat species was run effectively, clarifying five unique PCR fragments. These PCR sections compared the particular sizes expected for the five designated species. The results showed successful amplification of the target cyt b gene sequences with the expected amplicon sizes (271pb) for cattle, (274pb) for sheep, and (808pb) for dog meat. Amplification of the target mt DNA, and 12S rRNA-tRNA Val gene sequences with the expected amplicon sizes (359pb) for equine, and (290bp) for pork’s meat. The developed multiplex PCR assay was sensitive enough to detect 0.5% (w/w) adulterated meat under mixed matrices. It was concluded that the multiplex PCR could greatly minimize the cost for detection of meat adulteration