To test infrageneric classification and species delimitation within the pantropical moss genus Taxithelium (Pylaisiadelphaceae), we constructed a molecular phylogeny using three chloroplast loci (trnL, psbT and rps4), three mitochondrial loci (rps3, nad5 and nad4–5) and the nuclear marker ho1. Analyses of each locus separately and in various combinations, all support the monophyly of Taxithelium. Two major clades corresponding to taxonomically recognized subgenera were resolved within the genus. The first clade is composed of at least five smaller groups, four of which only include Southeast Asian plants and one is from the Americas and Africa; the latter is nested within the Southeast Asian groups. The second group appears to have a Southeast Asian origin with two dispersal events into America. Taxithelium is highly variable morphologically and includes plants with pluripapillose leaf cells as well as plants that lack papillae. Our data show that species T. nepalense, T. leptosigmatum, T. concavum, T. instratum, T. lindbergii and T. isocladum are each demonstrably monophyletic. On the other hand, T. planum and T. kerianum as circumscribed today are polyphyletic. The ho1 nuclear locus is used for the first time, with promising results for moss phylogenetic investigation.