While examining herbarium specimens of Trithuria inconspicua Cheeseman, we observed differences in the stigmatic hairs among plants from New Zealand’s North and South Islands. This motivated us to assess genetic and morphological variation within this species and its sister T. filamentosa Rodway from Tasmania. Samples were collected from lakes in the three disjunct geographic areas where the two species occur. Genetic variation in both species was assessed with simple sequence-repeat (SSR, microsatellite) markers and analyses of genetic distances. We also compared the morphology of northern and southern New Zealand T. inconspicua using fresh material. Samples of each species clustered together in a minimum evolution tree built from genetic distances. Trithuria filamentosa had more genetic diversity than did T. inconspicua. Within T. inconspicua, plants from lakes in the North Island and the South Island formed discrete genetic groups diagnosable by subtle morphological differences. Low levels of heterozygosity in both species are consistent with a high level of selfing, as suggested for other co-sexual Trithuria species, but unusual for a putative apomict. On the basis of genetic and morphological variation, we propose recognition of the northern New Zealand and southern New Zealand lineages of T. inconspicua at subspecies rank.