Abstract: A polymerase chain reaction (PCR) assay, for detection of ruminant-derived contaminants in processed animal feed was developed. The mitochondrial cytochrome-b (mtcyt-b) gene was used as the target DNA for PCR amplification. For detection of ruminant-specific mtcyt-b gene, a nested PCR assay was used in two amplification steps. First a 735-bp product was amplified using an outer primer pair (RSL1&RSR4), then a second amplification using nested (internal) primer pairs (RSL1&RSR3) produced a 483-bp product. The sensitivity of this nested PCR-based assay was found to be 10 fg (equivalent to 1000 copies) of mtcyt-b gene, extracted from ruminant whole blood. The PCR products were easily identified by size difference following visualization on an ethidium bromide-stained agarose gel. The described nested PCR assay could be used as simple and rapid methods for detection of ruminant-derived contaminants in animal feed.