Bovine spongiform encephalopathy, first identified in the UK in 1986, may have arisen from feeding scrapie infected meat-and-bone meal (MBM) to cattle that was produced under sub-optimal conditions. For public health and disease prevention reasons, banning the application of MBM in ruminant’s feeds was necessary. This study utilized a polymerase chain reaction (PCR) assay to identify bovine, porcine, ovine, and chicken MBM in animal diets. Bone fragments from feed samples were separated by chloroform separation. A nucleospin column was used for DNA extraction. Four pairs of primers that targeted at highly conserved regions of mitochondrial DNA (mtDNA) were employed in a PCR procedure to detect bovine, porcine, ovine and chicken DNA sequences. These gene fragments at the targeting region for the four species were 271, 225, 212 and 266 bps in size, respectively. In order to confirm the specific amplification of expected products from MBM for each species, the PCR products were digested with restriction enzymes HphI, MnlI, SspI and HindIII, and different length polymorphisms were observed. In the sensitivity assays, concentration as low as 1% of MBM in animal feeds could be detected by the PCR procedure. Based on our prevalence survey for commercial MBM in Taiwan, it predominantly consisted of bovine or porcine origins. This molecular approach provides a quick, highly reproducible and sensitive method to detect the MBM in feeds.