The trapdoor spider family Nemesiidae comprises 14 genera in Australia, the majority of which are included in the subfamily Anaminae. Here we provide evidence from a multigene molecular analysis of most Australian genera of Anaminae for a previously unrecognised clade that also differs from its sister-genus, Aname L. Koch, by the lack of a prominent asetose ventral depression on the pedipalpal tibia and the medium-sized mating spur on tibia I of males. This depression is a characteristic of all species of Aname examined to date, and represents a newly recognised character system in the subfamily. The new genus, named Hesperonatalius, is represented by three new species – H. maxwelli, sp. nov., H. harrietae, sp. nov. and H. langlandsi, sp. nov. – all from arid Western Australia.