Members of the Neobisiidae are currently classified in two subfamilies, Neobisiinae and Microcreagrinae. Taxonomic assignment to subfamily is based upon two morphological characters, neither of which is consistently found within either subfamily. The form of the galeae is elongate and hyaline in the Microcreagrinae, but reduced and sclerotic in the Neobisiinae. However, some members of the Microcreagrinae also have reduced galeae. The position of trichobothrium ist located on the fixed finger of the pedipalp chelae is generally positioned subdistally and closer to trichobothrium est in Neobisiinae but sub-basally and closer to trichobothrium ib in Microcreagrinae. However, members of the genus Parobisium, currently assigned to the subfamily Neobisiinae, have a microcreagrine-like subbasal trichobothrium ist. Since neither subfamily is defined by an undisputed apomorphy, the monophyly of both groups has long been questioned. In this study, we tested whether or not the two subfamilies are monophyletic by inferring the phylogeny of the family using DNA sequence data from the mitochondrial protein-coding gene, COI, and the nuclear ribosomal gene 28S. Results of the molecular phylogenetic analyses indicate that neither of the subfamilies is monophyletic as presently defined. We transfer the genus Parobisium to the Microcreagrinae in order to simultaneously obtain a monophyletic Neobisiinae and resolve character inconsistency for the position of trichobothrium ist, which is sub-distal in all Neobisiinae taxa included in our study. We also find that reduction of the galea is not a reliable character state at the subfamily level, and has occurred at least three times independently within the family.