Diatraea spp. (Lepidoptera: Crambidae) are a group of insects considered as an agriculture pest in many economically relevant crops such as sugarcane, sorghum, corn and rice. Currently, identification is based on the male genitalia. However, the availability of specimens collected from field and subjectivity on the character recognition can seriously hamper species identification, and therefore result in inadequate pest management. We described a protocol based on a fragment of the cytochrome oxidase subunit II (CO II) mitochondrial gene and Single-Strand Conformation Polymorphism (SSCP) methodology to properly recognizing each species and in consequence is proposed as an effective tool in Diatraea species identification.