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Food safety and forensic applications rely seriously on identifying meat samples of unknown species. Mutton meat is delicious and high-priced meat. Its tallow is a flavoring precursor to being authenticated by using different meat species. Mutton authenticity by beef meat that is genetically related to each other makes it difficult to detect fraud of products, mainly if the substitution is applied with fat, not only meat. So, accurate methods for detection must be applied to evaluate meat fraud based not only on protein but also on fat substitution. This work aimed to evaluate the mutton meat substitution by meat and fat of beef using multiplex PCR and Gas Chromatography-Tandem Mass Spectrometry (GC/MS/MS). Multiplex PCR detects cytochrome b in mutton meat adulterated with different concentrations of beef meat, even with 0.5%. PCR products amplified at 274 bp for mutton and 271 bp for beef. Fatty acid profile of pure mutton meat (tallow) samples and mutton authenticated with beef meat by GC/MS/MS. It was found that it contained 39.48 mg/100 g of total saturated fatty acids (TSFA), 60.48 mg/100 g of total unsaturated fatty acids (TUSFA), and 3.4 mg/100 g of trans-fatty acids (TFA). While after being authenticated with beef, these results changed to 49.58, 49.57, and 6.37 mg/100gm for TSFA, TUSFA, and TFA, respectively. In conclusion, Multiplex PCR and GC/MS/MS evidenced to be accurate and applicable for the recognition of mutton meat authentication.