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The Mesembrinellidae (Diptera: Oestroidea) comprise a small group of strictly Neotropical calyptrate flies, with 36 described species. The group has often been treated as a subfamily of Calliphoridae, but there is growing evidence that it corresponds to a distinct Oestroidea lineage. Internal relationships have so far been addressed based only on morphology, with results lacking resolution and support. This is the first molecular phylogeny for the group, which is based on the analyses of 80 terminal taxa (22 mesembrinellid and 28 outgroup species) and 5 molecular markers (ITS2, 28S, COI, COII and 16S). Maximum‐parsimony, maximum‐likelihood and Bayesian inference methods were used, the latter two with partitioning strategies considering codon position and secondary structure information. Results corroborate the Mesembrinellidae as a monophyletic lineage inside Oestroidea. Three clades were consistently …