The choanoflagellates were included in Chrysophyceae until Hibberd, 1975. Recent molecular phylogenetic reconstruction of the internal relationships of choanoflagellates allows the polarization of character evolution within the clade. Large fragments of the nuclear SSU and LSU ribosomal RNA, alpha tubulin, and heat-shock protein 90 coding genes were used to resolve the internal relationships and character polarity within choanoflagellates. Each of the four genes showed similar results independently and analysis of the combined data set (concatenated) along with sequences from other closely related species (animals and fungi) demonstrate that choanoflagellates are strongly supported as monophyletic and confirm their position as the closest known unicellular living relative of animals.