Chromosome analysis by pulsed-field gel electrophoresis shows a high level of genetic heterogeneity between the two subspecies of the green sulfur bacteria Chlorobium limicola analysed: C. limicola and C. limicola f. s. thiosulfatophilum. Currently, they are differentiated only by the ability to utilize thiosulfate as photosynthetic electron donor, and by their %G + C content (51% and 58.1%, respectively). However, the capacity to utilize thiosulfate as photosynthetic electron donor does not appear to be a useful criterion to differentiate between some strains of this species, because this jability is encoded by plasmids that are different depending on the thiosulfatophilum strain analysed. In contrast, this study reveals that the comparison of chromosomal restriction patterns is very useful as an additional aid for the differentiation and identification of C. limicola strains.