Background: Asini Corii Colla (ACC) (namely donkey hide gelatin, E'jiao in Chinese) was one of the most valuable tonic traditional Chinese medicines which is an infallible remedy to promote hematopoiesis. It should be produced by fresh or dried donkey hide according to Chinese Pharmacopeia (2015 edition) with a long-time decoction, while as donkey and horse (or mule) all belong to equids so their hides or their hide gelatins are share much in common, that cause the difficult in distinguishing raw materials donkey hide from horse/mule hide for manufacturer, and the challenge in the quality evaluation of ACC for regulatory authority to identify the adulterated with horse hide. Objective: To establish an effective quality evaluation methods for ACC focused on the qualitative-based identification of the raw material's authenticity, mainly to identify the species origin of the gelatins. Materials and Methods: DNA extracted from (1) Raw materials (hides of donkey, horse, mule, bovine and pig); (2) Five hide-glues (bovine, pig, donkey, horse and mule hide-glue); (3) 11 batches of ACC commercial products made by different manufactures from local drug stores. Polymerase chain reaction (PCR) method with newly designed horse-specific primers I and primer pair II. Results: Use the primer pair I, a 234 bp target product could be amplified sensitively from the DNA sample of horse/mule adulterated commercial ACC products, though the DNA in commercial products is severely degraded. A 219 bp product could be amplified specifically from the DNA sample of horse/mule hide, while the results were all negative for the DNA templates of donkey hide, its gelatin and ACC products without adulteration. Conclusion: The developed PCR method based on primer I and II provide an effective approach to identify the species origin of highly processed product ACC (primer pair I) as well as to distinguish the raw material donkey hide (primer pair II), which might enlighten a new strategy to the Quality Evaluation of ACC. Abbreviations Used: ACC: Asini Corii Colla; TCMs: Traditional Chinese Medicines; SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis; IEF: Isoelectric focusing; GFC: Gel filtration chromatography; 2-DE: Two-dimensional electrophoresis; PCR: Polymerase chain reaction. © 2017 Pharmacognosy Research Published by Wolters Kluwer Medknow.