The genetic diversity and relationships of 81 Rhodothermus isolates from different geothermal environments in Iceland were examined by analysis of electrophoretically demonstrable allelic variation of 13 genes encoding enzymes. All the enzymes were polymorphic. A total of 71 distinctive multilocus genotypes (electrophoretic types, ETs) were identified. The mean genetic diversity per locus (H1) was 0.586. The relatively high genetic variance observed within Rhodothermus isolates from different locations is most likely the result of genetic changes occurring independently in the locations studied. A high Gst value (0.284) indicates that a considerable part of the variance observed is due to differences between locations. Cluster analysis revealed two major groups of ET clusters diverging at a genetic distance of 0.75, reflecting strongly the geographic origin of isolates. Estimation of the association index (I(A)) indicates that Rhodothermus marinus is a clonal species in which recombination events occur rarely. Partial or whole sequencing of the 16S rRNA genes of Rhodothermus isolates grouping at genetic distance of 0.40 confirmed that all the isolates belonged to the species Rhodothermus marinus. The results of this study confirm that, despite phylogenetic and phenotypic similarity, genetic diversity within Rhodothermus marinus is quite high.