In Brazil, the species Diatraea flavipennella and D. saccharalis play an important role in the sugar and alcohol agribusiness by causing many damages in sugarcane fields. The egg, larva, pupa, and adult stages are very morphologically similar between these species, and the identification can be confused. The internal transcribed spacer 2 (ITS 2) from ribosomal DNA has important features as evolutionary divergence. It is a good marker for species identification, participates in the rDNA processing, and has been applied in phylogenetic and population studies. This study aimed to make available a molecular marker to assist on the identification method of pests’ species of Diatraea and to identify possible traces of Cotesia in the resistant host. The DNA was extracted from the egg, larva, and adult samples. PCR amplicons were purified and sequenced. The sequences were analyzed in MEGA 5.01. The ITS 2 length was 410 bp in D. flavipennella and 448 bp in D. saccharalis. The GC content was similar between the species. Three microsatellite loci were present in D. saccharalis and absent in D. flavipennella, contributing to differences in ITS 2 length in the species. An additional 367-bp band was attributed to Cotesia spp. The differences among ITS 2 from D. flavipennella, D. saccharalis, and Cotesia sp were sufficient to identify them on electrophoresis gel and sequencing. The presence of Cotesia sp traits in adult D. flavipennella showed possible host refractoriness, but further studies are necessary.