Rhizoma Curcumae (Ezhu) is a traditional Chinese medicine that has been used in removing blood stasis and alleviating pain for over a thousand years. Three species of Curcuma rhizomes are being used, which include Curcuma wenyujin, Curcuma phaeocaulis, and Curcuma kwangsiensis. In China, the production of Rhizoma Curcumae largely depends on agricultural farming. The essential oils are considered as active constituents in Rhizoma Curcumae, which include curdione, curcumol, and germacrone. On the basis of the yield of curdione, curcumol, and germacrone in an orthogonal array design, the optimized extraction condition was developed. The amounts of these compounds within essential oils in Rhizoma Curcumae varied according to different species and their regions of cultivation. Chemical fingerprints were generated from different species of Curcuma, which therefore could serve as identification markers. In molecular genetic identification of Rhizoma Curcumae, the 5S-rRNA spacer domains of 5 Curcuma species, including the common adulterants of this herb, were amplified, and their nucleotide sequences were determined. Diversity in DNA sequences among various species was found in their 5S-rRNA spacer domains. Thus, the chemical fingerprint together with the genetic distinction could serve as markers for quality control of Curcuma species.