In Ayurveda and Thai traditional medicines, material from Coscinium fenestratum is commonly prescribed as active ingredients with diverse therapeutic purposes. However, C. fenestratum is also a seriously endangered medicinal liana. Thus, its crude material is very rare and is being substituted with substances from Arcangelisia flava or Fibraurea tinctoria (Menispermaceae), which have high morphological similarity. In this current study, nuclear 18S ribosomal RNA (rRNA) gene and nuclear ribosomal DNA internal transcribed spacer (ITS) gene sequences with the polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPs) technique were exploited to identify these three species. The nuclear 18S rRNA gene sequences of C. fenestratum, A. flava, and F. tinctoria consisted of 1809, 1805, and 1809 base pairs (bps), respectively, while their ITS gene regions were 694, 622, and 631 bps in length, respectively. The 18S rRNA gene of C. fenestratum digested with SmaI restriction enzyme displayed the electrophoresis profile of 729 and 790 bps; for A. flava and F. tinctoria, the digested products showed fragments of 1519 bps. Although the ITS gene regions of A. flava and F. tinctoria had unrecognized sequences with SalI, the SalI-digested ITS of C. fenestratum exhibited fragments of approximately 599 bp. Thus, the 18S rRNA gene and ITS gene sequences with PCR-RFLPs were proven to be powerful molecular markers for identifying C. fenestratum and distinguishing it from the other two Menispermaceae plants.