Negativicutesis currently divided into one order and two families on the basis of 16S rRNA gene sequence phylogenies. We report here comprehensive comparative genomic analyses of the sequenced members of the class Negativicutesto demarcate its different evolutionary groups in molecular terms, independently of phylogenetic trees. Our comparative genomic analyses have identified 14 conserved signature indels (CSIs) and 48 conserved signature proteins (CSPs) that either are specific for the entire class or differentiate four main groups within the class. Two CSIs and nine CSPs are shared uniquely by all or most members of the class Negativicutes, distinguishing this class from all other sequenced members of the phylum Firmicutes. Four other CSIs and six CSPs were specific characteristics of the family Acidaminococcaceae, two CSIs and four CSPs were uniquely present in the family Veillonellaceae, six CSIs and eight CSPs were found only in Selenomonasand related genera, and 17 CSPs were identified uniquely in Sporomusaand related genera. Four additional CSPs support a pairing of the groups containing the genera Selenomonasand Sporomusa. We also report detailed phylogenetic analyses for the Negativicutesbased on core protein sequences and 16S rRNA gene sequences, which strongly support the four main groups identified by CSIs and by CSPs. Based on the results from different lines of investigation, we propose a division of the class Negativicutesinto an emended order Selenomonadalescontaining the new families Selenomonadaceaefam. nov. and Sporomusaceaefam. nov. and two new orders, Acidaminococcalesord. nov. and Veillonellalesord. nov., respectively containing the families Acidaminococcaceaeand Veillonellaceae.