5S ribosomal RNA (rRNA) genes are known to move in and out of various tandemly repeated eukaryotic gene families during evolution. Here, we investigated the organization of 5S rRNA genes linked to the ribosomal DNA (rDNA) units in 147 fungal species using available sequence and genome databanks. Whereas some fungal species have no 5S rRNA genes in their rDNA units, others have one or two 5S rRNA copies linked on the same or the opposite strand. There were at least 13 independent changes during the evolution of fungal species. These include two 5S rRNA genes loss, five 5S rRNA genes inversions and six 5S rRNA genes insertions (including duplications). The lower frequency of 5S rRNA genes loss might be due to the fact that these events are more likely to affect fitness. The maximum time required for 5S rRNA gene organization to change between related species was estimated to be 7.5 millions years based on the sequences of the elongation factor alpha genes of Candida glabrata and Saccharomyces mikatae. This time is much longer than the homogenization time predicted from theoretical and experimental studies and likely reflects the lack of closely related species or strains in our data set.