EvaGreen multiplex real-time polymerase chain reaction (EMRT-PCR) was designed for an assay that can join the advantages of multiplex PCR and real-time PCR to recognize animal genes more quickly in pet foods. EMRT-PCR based on melting temperatures discrimination by using EvaGreen fluorescence dye was developed for the analysis of pork and poultry in pet food. The method combines the use of poultry- and pork-specific primers that amplify small fragments of 12S rRNA and mitochondrial DNA genes. Appropriate mixtures of poultry and pork meat in reference samples were used to develop the assay. Gene yields of poultry and pork were represented in two melting peaks generated simultaneously at temperatures of 80.5 and 87.2 A degrees C, respectively. Based upon the assay results, it has been concluded that EMRT-PCR assay might be an efficient tool for the verification of species origin in pet foods.