The non-transcribed spacer of the ribosomal DNA repeat of Pythium pachycaule was found to exist in two versions, one about 200 bp longer than the other. Both versions contained a conserved 5s rRNA gene in inverted orientation and a diverged copy of the 5s sequence as a tandem repeat, with a spacer of about 180 bp between them. This is the first report of multiple 5s-like sequences in the non-transcribed spacer of the rDNA of any organism. The two diverged 5s sequences differ from each other at nine positions in the 5' half of the sequence, but they have the same central 10-bp deletion and their 3' halves are identical. The diverged sequences, 5s' and 5s'', when aligned with the 5s sequence, have the same base at 63.9 and 58.8% of the positions respectively. The spacers between 5s sequences in both versions were highly conserved. The secondary structures of potential rRNA products of the diverged 5s sequences indicate that they may be pseudogenes. The relationships between the functional 5s gene and the diverged gene copies suggest that the 5s sequence family of P. pachycaule originated by duplication of an ancestral gene, followed by divergence and the introduction of heterogeneity. This work shows the potential for tandemization of 5s genes in the NTS, and may shed light on the evolutionary events that led to the transition from the rDNA-linked 5s genes found in Pythium species with filamentous zoosporangia to the unlinked tandem arrays found in Pythium species with globose zoosporangia.