The description of materials and methods used in this study addresses sample preparation and DNA extraction, rDNA IGS amplification, PCR products cloning and sequencing, sequence confirmation, and sequence analysis. The experiment found that the repeat sequence structure of rDNA IGS of cannabis sativa was similar to the reported structure of other plant rDNA IGS; however, the sequence of this locus for cannabis sativa was significantly different from that of any other plant. The typical transcription initiation site (TIS), TATA(G)TA(N)GGGG, could not be determined by comparison with other reported sequences; therefore, it will be necessary to determine the exact TIS by S1 nuclease mapping. Another possibility is that this is a nonfunctional pseudogene, so the TIS was not observed. These specific and complex variations of IGS may be related to the species and geographic distributions of plants. The sequence structure of rDNA IGS of cannabis sativa provides a potential means of classification and identification among different cultivars and even within individual plants, making it a valuable locus in the establishment of the population data of different accessions or origins of cannabis. 2 figures, 1 table, and 30 references