The circumscription of the Plagiotheciaceae was evaluated cladistically, using anatomical and morphological data, plus rps4 and trnL-trnF gene sequences. Two analyses were conducted with 37 ingroup species, representing 14 genera, and eight outgroup species. First, the rps4 (73 informative nucleotide sites) plus trnL-trnF data set (39 sites) was analyzed alone. This initial analysis suggests a highly polyphyletic Plagiotheciaceae, with Acrocladium, Catagonium, Herzogiella, Isopterygiopsis pulchella (Hedw.) Iwats., Isopterygium, Plagiothecium piliferum (Sw.) Schimp., Pseudotaxiphyllum elegans (Brid.) Iwats., P. pohliaecarpum (Sull. & Lesq.) Iwats., Rhizofabronia, Struckia, and Taxiphyllum placed among the outgroup taxa. In a second analysis, the rps4 trnL-trnF data set was combined with anatomical and morphological data (adding 53 informative characters). The results of the latter analysis are congruent with the first analysis regarding Herzogiella adscendens (Lindb.) Iwats., Isopterygium, and Taxiphyllum, but resolves the rest of the Plagiotheciaceae as a monophyletic group. The results suggest that the following genera belong to the family: Acrocladium, Bardunovia, Catagonium, Herzogiella, Isopterygiopsis, Orthothecium, Myurella, Plagiothecium, Platydictya, Pseudotaxiphyllum, Rhizofabronia, and Struckia. DNA sequence, morphological, and anatomical data are all important when evaluating the circumscription and internal relationships of the family. We suggest that especially the relationships among Plagiothecium, Pseudotaxiphyllum, and Struckia species can be better resolved by additional molecular evidence. The ancestor of the Plagiotheciaceae was most likely a southern temperate species, whereas most of the later diversification within the family occurred in the northern temperate zone. Only one genus, Rhizofabronia, evolved in tropical mountains, whereas scattered members of other genera occur in similar environments. Adaptations for calcareous substrates evolved either in the ancestor of the entire Isopterygiopsis-Plagiothecium clade and were lost twice, or they evolved independently in Isopterygiopsis and in a clade consisting of Bardunovia, Myurella, Orthothecium, and Platydictya.