tRNA(Leu) UAA (trnL) intron sequences are used as genetic markers for differentiating cyanobacteria and for constructing phylogenies, since the introns are thought to be more variable among close relatives than is the 16S rRNA gene, the conventional phylogenetic marker. The evolution of trnL intron sequences and their utility as a phylogenetic marker were analysed among heterocystous cyanobacteria with maximum-parsimony, maximum-likelihood and Bayesian inference by comparing their evolutionary information to that of the 16S rRNA gene. Trees inferred from the 16S rRNA gene and the distribution of two repeat classes in the P6b stem-loop of the trnL intron were in clear conflict. The results show that, while similar heptanucleotide repeat classes I and II in the P6b stem-loop of the trnL intron could be found among distant relatives, some close relatives harboured different repeat classes with a high sequence difference. Moreover, heptanucleotide repeat class II and other sequences from the P6b stem-loop of the trnL intron interrupted several other intergenic regions in the genomes of heterocystous cyanobacteria. Cluster analyses based on conserved intron sequences without loops P6b, P9 and parts of P5 corresponded in most clades to the 16S rRNA gene phylogeny, although the relationships were not resolved well, according to low bootstrap support. Thus, the hypervariable loop sequences of the trnL intron, especially the P6b stem-loop, cannot be used for phylogenetic analysis and conclusions cannot be drawn about species relationships on the basis of these elements. Evolutionary scenarios are discussed considering the origin of the repeats.