This study presents the first phylogenetic estimate for the pipunculid subfamily Chalarinae (genera Chalarus Walker, Jassidophaga Aczél and Verrallia Mik) based on an analysis of one mitochondrial coding (cytochrome oxidase 1) and two nuclear non-coding genes (28s and ITS2), using parsimony under direct optimization as implemented in poy4. It completes earlier taxonomic works on these groups. The voucher material used was primarily of Palaearctic origin. The study strongly supports the monophyletic origin of Chalarinae as well as of Chalarus and Verrallia, but failed to recover a monophyletic lineage for Jassidophaga. Whereas the taxa of Jassidophaga with predominantly black-coloured legs clustered as a monophyletic sister to Verrallia, an Oriental Jassidophaga species with predominantly yellow/light brown-coloured legs represents a distinct genetic lineage. The Chalarus species included were resolved into eight well-supported genetic clades: C. angustifrons group, C. basalis group, C. clarus lineage, C. holosericeus group, C. immanis lineage, C. indistinctus group, C. latifrons group and C. spurius group. A phenetic analysis focused on intra- and interspecific genetic distances within the subfamily. As a consequence, the eastern Palaearctic C. rectifrons Morakote is proposed as a junior synonym of C. angustifrons Morakote (syn.n.). The structure of the C. basalis species group was investigated further with the intersimple sequence repeat (ISSR) primer (GACA) ₄.