Cane borer, Diatraea spp., with associated natural enemies were collected at different municipalities of the State of Guanajuato, México, and identified morphologically and molecularly. Morphological identification compared characteristics of male genitalia, while molecular identification was achieved by gene sequencing of subunit I of the mitochondrial DNA enzyme cytochrome c oxidase. Entomopathogenic fungi from larvae collected in the field were identified molecularly using internal transcribed spacers (ITS) of ribosomal DNA. The species of cane borer identified morphologically and molecularly was southwestern corn borer, Diatraea grandiosella Dyar. A maximum likelihood tree grouped the sequences of the species D. grandiosella with 99% confidence, corroborating identification of the species. Nucleotide sequences confirmed the identity of Beauveria bassiana (Balsamo) Vuillemin⁵ and Metarizhium rileyi (Farlow) Kleper⁵ entomopathogenic fungi with 100% confidence. Two parasitoids and one predator of D. grandiosella larvae collected were Digonogastra kimballi Kircland⁶ and Chrysoexorista lineata Wolp⁷, and Enoclerus bombicinus Chevrolat⁸, respectively.