Entactinaria, an order of Radiolaria, are defined by a specific skeletal structure called "initial spicular system (ISS)". The oldest entactinarians appeared in the Ordovician period, and the extant species are thought to have survived until today. However, the morphological observation revealed that the ISSs of entactinarian families are highly variable, and the validity of this characteristic is questionable. This is supported by the results of 18S and 28S rRNA molecular phylogenetic analysis that suggested the polyphyly of the four families analyzed in this study. Orosphaeridae, Rhizosphaeridae, Hexalonchidae and Hexastylidae should be excluded from the order Entactinaria. Considering the present results and the diversity in the ISS, it is also possible that "living entactinarians" are polyphyletic.