Pastirma is a high value, traditional, dry cured, and edible coated meat product. For economic and sociocultural reasons, it is important to know which meat species are used in pastirma. The present study aims to carry out the determination of the meat species in pastirma. A total of 144 pastirma samples were collected from different stores. After genomic DNA isolation, the total DNAs obtained were subjected to polymerase chain reaction (PCR) using specially designed novel primers amplifying the mitochondrial ND4 (MT-ND4) gene region, specific for cattle (Bos taurus), water buffalo (Bubalus bubalis), horse (Equus caballus), and donkey (Equus asinus) species. For the identification of the meat species, gel purified 952 bp PCR products were digested with fast digest SaqAI restriction enzymes selected based on preliminary in silico analyses. The results of the present study revealed that all of the pastirma samples were made from cattle meat. Cross-reactions and false-positive identifications are serious problems in routine determination of the meat species in food control laboratories. It is important to perform fast, inexpensive determination with high sensitivity and specificity for the identification of meat species. This study has shown that meat species can be detected with high specificity in products such as pastirma, which uses large pieces of meat cuts.