The subfamily Aleocharinae Fleming is one of the largest and most diverse lineages of staphylinid beetles (≈12,000 species). Being one of the most taxonomically difficult groups of beetles, the classification of the 52 tribes in this subfamily is artificial and almost void of any phylogenetic context. As a preliminary step toward building a tribal-level phylogeny for this lineage of beetles, a molecular investigation of partial sequences of 12s and 16s mitochondrial genes was undertaken for 21 species in eight different tribes. These sequences were analyzed using parsimony, bayesian, and maximum likelihood methods. Results largely support the findings of previous morphological analyses. The “higher” Aleocharinae form a monophyletic group. The tribe Myllaenini Ganglbauer is supported as a member of the higher Aleocharinae. The subtribe Gyrophaenina Kraatz is monophyletic. These results suggest that 12s and 16s mitochondrial genes are phylogenetically informative molecular characters for building a tribal-level phylogeny of the Aleocharinae.