Pheretima is an animal-derived traditional Chinese medicines (TCMs). The chemical quality markers of Pheretima used to distinguish different species are still ambiguous. Under this premise, a strategy integrated DNA barcoding with metabolomics is promoted for identifying Pheretima and screening distinguishable combinatorial chemical quality marker (DCQ-marker) between Pheretima aspergillum (P. aspergillum) and Pheretima vulgaris Chen (P. vulgaris). As a result, adenosine, adenine, L-phenylalanine and uridine are successfully selected as DCQ-markers between P. aspergillum and P. vulgaris. This study provides convenient strategy for quickly screening DCQ-marker between P. aspergillum and P. vulgaris. It will be meaningful for further promoting quality control on Pheretima and providing a reference for the quality evaluation of other animal-derived TCMs.