The phylogeny of the subfamily Sciarinae is reconstructed by the result of maximum likelihood, maximum parsimony, and Bayesian inference of 4,809 bp from two mitochondrial (COI and 16S) and two nuclear (18S and 28S) genes. The current subfamily Sciarinae is polyphyletic, since the genera Chaetosciara, Mouffetina, Schwenckfeldina, and Scythropochroa are grouped separately as the sister clade of other subfamilies. The monophyly of the genera Chaetosciara, Sciara, Scythropochroa, and Trichosia s. str. is strongly supported; however, the genus Leptosciarella s. l. is polyphyletic due to the separation of subgenus Leptosciarella (Leptospina) as a sister taxa of remaining Sciarinae (Leptosciarella s. str., Sciara, and Trichosia s. str.). The classification of the Sciarinae is revised and discussed on the basis of molecular phylogenetic hypotheses.