The infrafamilial relationships of Podostemaceae were deduced from nucleotide sequences of the chloroplast matK gene. The matK phylogenetic analyses show that Podostemaceae are composed of two major clades that correspond to the subfamily Tristichoideae sensu stricto and Weddellina and the subfamily Podostemoideae. Weddellina, which has long been recognized as a member of the Tristichoideae, is sister to the Podostemoideae, supporting the classification that recognized a third subfamily Weddellinoideae. Malaccotristicha malayana and Terniopsis sessilis form a basal clade in Tristichoideae sensu stricto. Tristichoideae show a high morphological diversity and, surprisingly, a close relationship exists between Dalzellia zeylanica and Indotristicha ramosissima, which remarkably differ in their body plans. A few genera defined by particular characters, such as Synstylis and Torrenticola, merge into clades of other larger genera. The Podostemoideae taxa studied are composed of two American clades, an Asian‐Australian clade and a Madagascan clade, and may suggest that the subfamily perhaps originated in America and migrated to the Old World.