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The bivalve superfamily Lucinoidea is usually considered to comprise six separate families: Lucinidae, Thyasiridae, Ungulinidae, Fimbriidae, Mactromyidae and Cyrenoididae. Chemoautotrophic chemosymbiosis with sulphide-oxidizing bacteria is present in all studied species of Lucinidae, Fimbriidae and many, but not all, Thyasiridae. However, it is absent from Ungulinidae. The Mactromyidae are likely to be an entirely fossil group with doubtful affinities with Lucinoidea. The Cyrenoididae are poorly investigated, but anatomical features suggest they are unrelated to lucinoids. To investigate phylogenetic relationships within the Lucinoidea and test hypotheses concerning the evolution of the chemosymbiosis, a molecular study was made using sequences of 18S and 28S rRNA genes. The study incorporated species of Ungulinidae (two genera, two species), Thyasiridae (three species), Fimbriidae (one species) and many Lucinidae (31 species, 19 genera) as well as a range of outgroups representing major groups of heterodont and palaeoheterodont bivalves. The results demonstrate that the monophyly of the Lucinoidea is not supported. The Ungulinidae and Thyasiridae are unrelated to the Lucinidae. Ungulina and Diplodonta of the Ungulinidae group with a clade comprising Veneroidea, Arcticoidea and Mactroidea. The three Thyasira species analysed form a monophyletic branch in a basal position among the heterodont bivalves. The only member of the Fimbriidae examined, Fimbria fimbriata, groups within the Lucinidae and separation as a family is not supported. The Lucinidae form a monophyletic group within which several distinct and well-supported clades and lineages are recognized: the Myrtea clade, the ‘Anodontia’ clade, Fimbria lineage, Phacoides pectinatus lineage, and two clades comprising all other lucinids. The implication of non-monophyly of the superfamily Lucinoidea is that Thyasiridae represent an independent acquisition of bacterial chemosymbiosis and this is reflected in major morphological differences from the Lucinidae.