Ephedrae herba has been used in traditional Chinese and Japanese (Kampo) medicine from ancient times, with the primary resource being in China. In the present study, a field survey as well as molecular and chemical assessments were conducted on Ephedra plants in Mongolia to clarify whether they could be an alternative resource of the Ephedrae herba used in Japanese Kampo medicine. Ephedra sinica, E. equisetina, E. przewalskii, E. regeliana, E. monosperma and an unknown taxon (ESP) collected in Mongolia were divided into 9 genotypes on the basis of nucleotide sequences of 18S ribosomal RNA (rRNA) gene and trnK gene. E. sinica, E. equisetina, and E. monosperma presented completely identical sequences to the corresponding species from China. The sequences of trnK gene and 18S rRNA gene provide a useful index for identification and taxonomic classification of Mongolian Ephedra plants. Quantitative analysis of 5 ephedrine alkaloids revealed that almost all Mongolian Ephedra plants contained high amounts of total ephedrine alkaloids (TAs, 1.86-4.90%) and a high percentage of pseudoephedrine in TAs differed obviously from the Chinese. E. sinica and E. equisetina found in eastern and central Mongolia, showing total contents of ephedrine and pseudoephedrine higher than 1.43%, were potential new resources of Japanese Pharmacopoeia grade Ephedrae herba.