Two of the largest subfamilies of Crambidae, Pyraustinae and Spilomelinae, have 341 species recorded in Japan, but their natural relationships are poorly understood. We reconstructed a molecular phylogeny based on one mitochondrial (COI) and three nuclear genes (CAD, EF1-α, RpS5) for 129 species in 69 genera of Spilomelinae and 62 species in 27 genera of Pyraustinae from Japan. The concatenated data was analyzed by maximum likelihood and Bayesian inference methods. Our results recovered most tribes recognized in a previous study, but Pyraustini is paraphyletic as it includes Portentomorphini, and the paraphyletic Nomophilini comprise Trichaeini. The species-rich Spilomelinae genera Patania, Nacoleia, Glyphodes, and Syllepte are polyphyletic and need thorough revision. In Pyraustinae, a “Monocotyledon-clade” and Anania sensu lato were recognized for the first time. Based on phylogenetic results and morphological investigations, we propose six taxonomic changes, including the transfer of Acropentias from Spilomelinae to Lathrotelinae and Mabra from Spilomelinae to Pyraustinae. Fourteen unplaced genera of Spilomelinae and two of Pyraustinae are newly assigned to tribes.