The rDNA intergenic spacer (IGS) structure of Cannabis sativa was established and can be used for classification and identification of this species. In this study, DNA fragments of rDNA IGS were amplified by PCR from Cannabis sativa plant extracts and a 1387 bp fragment was obtained. DNA sequence analysis revealed six different repeat motifs. In the middle of the IGS sequence, there were three sequence motifs, and the same three sections of DNA were then repeated with minor variation in sequence. The terminal region of the IGS was composed of another three different repeat units; multiple copies of these terminal repeat motifs were present in no discernible order. Within six repeat motifs, point variations were observed in five. The DNA sequence of the locus was compared with all the plant sequences registered in GenBank by the Fasta program of GCG software with the result that this DNA fragment was significantly different from any other DNA sequence recorded to date. The most similar sequence was that of Hops (Humulus lupulus), but with a similarity of only 88.9% over 579 bp. These specific and complex variations of IGS may be related to the species and geographic distributions.