The phylum "Chloroflexi" contains highly divergent groups of bacteria. To understand the evolutionary relationships among these bacteria, phylogenetic trees were constructed based upon concatenated sequences for 20 conserved proteins and comparative genomic analyses were carried out to identify molecular markers (conserved signature indels or CSIs) that are specific for different clades of Chloroflexi. In phylogenetic trees based upon either concatenated protein sequences or the 16S rRNA gene, species from the class Chloroflexi and the order Chloroflexales formed strongly supported clades. The species from these clades are also clearly distinguished from other bacteria based upon 5 and 9 identified CSIs, respectively, in important proteins that were specific for these clades. Additionally, three CSIs that were specific for the genus Chloroflexus and four CSIs specific for the genus Roseiflexus were also identified. In phylogenetic trees, the species Oscillochloris trichoides (family Oscillochloridaceae) formed a strongly supported clade with the species from the genus Chloroflexus (and with Chloronema in the 16S rRNA gene tree). A specific relationship of O. trichoides to the Chloroflexus spp. is also strongly supported by 7 CSIs that are uniquely shared by the species from these genera but not found in Roseiflexus or any other bacteria. In addition to their phylogenetic clustering and shared presence of many novel CSIs, the species from the genera Chloroflexus and Oscillochloris (and also Chloronema) also differ from species of the genera Roseiflexus (and Heliothrix) by their green color, shared presence of the chlorosomes and Bchl c (in addition to Bchl a and d in some species), by their fatty acid profiles, and by the presence of β- and γ-carotenes and quinone MK-10. Based upon these observations, we propose division of the order Chloroflexales into two suborders: the first of these suborders Chloroflexineae subord. nov. is comprised of the family Oscillochloridaceae (emended to include the genus Chloronema) and a new family Chloroflexaceae fam. nov. consisting of the genus Chloroflexus. The second suborder Roseiflexineae subord. nov. contains a new family Roseiflexaceae fam. nov. comprised of the genera Roseiflexus and Heliothrix; orange-red bacteria lacking chlorosomes and Bchl c and differing from the Chloroflexineae in their carotenoids, quinones and fatty acid profiles. Additionally, we also provide here formal descriptions of the class Chloroflexia class. nov. [corrected], and of the orders Chloroflexales ord. nov. and Herpetosiphonales ord. nov. Lastly, our phylogenetic and comparative analyses provide either no or very weak support for a grouping together of the different classes (viz. Chloroflexi, Thermomicrobia, Dehalococcoidetes, Anaerolineae, Caldilineae and Ktedonobacteria) that are currently part of the phylum Chloroflexi. However, a specific grouping of the classes Chloroflexi and Thermomicrobia (as well as 'Thermobaculum') is supported by both phylogenetic means and the identified CSIs. Based upon these results, it is suggested that the phylum Chloroflexi "sensu stricto" should be comprised only of the classes Chloroflexi and Thermomicrobia and the other four classes (viz. Dehalococcoidetes, Anaerolineae, Caldilineae and Ktedonobacteria), which are at present part of this "superphylum" should be regarded as taxa related to the phylum Chloroflexi "sensu stricto", awaiting more detailed investigations to clarify their relationships to each other and other phyla of bacteria.