The presence of shared conserved insertions or deletions in proteins (referred to as signature sequences) provides a powerful means to deduce the evolutionary relationships among prokaryotic organisms. This approach was used in the present work to deduce the branching orders of various eubacterial taxa consisting of photosynthetic organisms. For this purpose, portions of the Hsp60 and Hsp70 genes, covering known signature sequence regions, were PCR-amplified and sequenced from Heliobacterium chlorum, Chloroflexus aurantiacus and Chlorobium tepidum. This information was integrated with sequence data for several other proteins from numerous species to deduce the branching orders of different photosynthetic taxa. Based on signature sequences that are present in different proteins, it is possible to infer that the various eubacterial phyla evolved from a common ancestor in the following order: low G+C Gram-positive (H. chlorum ) → high G+C Gram-positive → Deinococcus–Thermus → green non-sulphur bacteria (Cf. aurantiacus ) → cyanobacteria → spirochaetes →Chlamydia–Cytophaga–Aquifex–flavobacteria–green sulphur bacteria (Cb. tepidum ) → proteobacteria (α, δ and ε) and → proteobacteria (β and γ). The members of the Heliobacteriaceae family that contain a Fe–S type of reaction centre (RC-1) and represent the sole photosynthetic phylum from the Gram-positive or monoderm group of prokaryotes are indicated to be the most ancestral of the photosynthetic lineages. Among the Gram-negative bacteria or diderm prokaryotes, green non-sulphur bacteria such as Cf. aurantiacus, which contains a pheophytin–quinone type of reaction centre (RC-2), are indicated to have evolved very early. Thus, the organisms containing either RC-1 or RC-2 existed before the evolution of cyanobacteria, which contain both these reaction centres to carry out oxygenic photosynthesis. The eubacterial divisions consisting of green sulphur bacteria and proteobacteria are indicated to have diverged after cyanobacteria. Some implications of these results concerning the origin of photosynthesis and the earliest prokaryotic fossils are discussed.