We amplified, TA-cloned, and sequenced the 16S-23S internal transcribed spacer (ITS) regions from single isolates of several cyanobacterial species, Calothrix parietina, Scytonema hyalinum, Coelodesmium wrangelii, Tolypothrix distorta, and a putative new genus (isolates SRS6 and SRS70), to investigate the potential of this DNA sequence for phylogenetic and population genetic studies. All isolates carried ITS regions containing the sequences coding for two tRNA molecules (tRNA and tRNA). We retrieved additional sequences without tRNA features from both C. parietina and S. hyalinum. Furthermore, in S. hyalinum, we found two of these non-tRNA-encoding regions to be identical in length but different in sequence. This is the first report of ITS regions from a single cyanobacterial isolate not only different in configuration, but also, within one configuration, different in sequence. The potential of the ITS region as a tool for studying molecular systematics and population genetics is significant, but the presence of multiple nonidentical rRNA operons poses problems. Multiple nonidentical rRNA operons may impact both studies that depend on comparisons of phylogenetically homologous sequences and those that employ restriction enzyme digests of PCR products. We review current knowledge of the numbers and kinds of 16S-23S ITS regions present across bacterial groups and plastids, and we discuss broad patterns congruent with higher-level systematics of prokaryotes.