AbstractInitiation of protein biosynthesis is an essential process occurring in cells throughout the three phylogenetic domains, Bacteria, Archaea, and Eucarya. IF1/eIF1A and IF2/eIF5B, two conserved translation initiation factors are involved in this important step of protein biosynthesis. The essentiality, universal distribution, conservation, and interspecies functional homology of both factors are a unique combination of properties ideal for molecular phylogenetic studies as demonstrated by the extensively compared SSU rRNAs. Here, we assess the use of IF1/eIF1A and IF2/eIF5B in universal and partial phylogenetic studies by comparison of sequence information from species within all three phylogenetic domains and among closely related strains of Haemophilus parainfluenzae . We conclude that the amino acid sequence of IF1/eIF1A‐IF2/eIF5B is a universal phylogenetic marker and that the nucleotide sequence of the IF2/eIF5B G‐domain is more credible than SSU rRNA for the construction of partial phylogenies among closely related species and strains. Because of these two application levels, IF1/eIF1AIF2/eIF5B is a phylogenetic “dual level” marker.