Asterella is a large group of chambered liverworts with a worldwide distribution; the genus is primarily defined by the presence of a pseudoperianth, a cage-like structure surrounding each capsule in the carpocephalum. Molecular phylogenies of a range of Asterella species were reconstructed using two chloroplast DNA sequences (ca. 70% of the matK coding region and the trnL intron), in order to study relationships of species within the genus and to test hypothetical relationships predicted by spore ornamentation types. The matK gene has not been used previously in bryophytes. Sixteen species of Asterella were sampled; representatives of the other four (smaller) genera of Aytoniaceae were also sampled i.e., Cryptomitrium (one species), Mannia (two species), Reboulia (one species), and Plagiochasma (two species). Outgroups selected from other families of Marchantiopsida were Athalamia (Cleveaceae), Lunularia (Lunulariaceae), Marchantia (Marchantiaceae), Riccia (Ricciaceae), and Targionia (Targioniaceae). Three analyses were performed: 1) matK, 2) trnL, and 3) a combined weighted matK/trnL analysis. The last analysis produced a single most parsimonious tree. Analyses rooted on Lunularia produced a stable resolved tree topology, with very strong support for Aytoniaceae as a monophyletic group, but with Asterella appearing as a paraphyletic genus including its four sister-genera. Paraphyly of ‘Asterella’ suggests that the pseudoperianth is actually a plesiomorphy within the family that has been lost during at least three separate evolutionary events. The major clades within Aytoniaceae have distinctive spore ornamentation types, and some of the infrageneric groups of Asterella inferred from spore characters are supported by the molecular analyses. The traditional subfamily groupings of genera within Aytoniaceae, however, are not supported. The taxonomic implications of the study are discussed.