The sugarcane borer, Diatraea saccharalis (Lepidoptera: Crambidae), is a stemborer which can cause significant economic damage in sugarcane throughout America. It is a widespread pest where sugarcane is cultivated and it has been managed through various methods including biological control. Due to its broad distribution, population genetics have been investigated to search for divergent populations or cryptic species. In North America, populations of larvae of D. saccharalis from Texas, Louisiana and Florida were collected and raised to adults. Adults were identified and compared using adult genitalia and by sequencing a region of the mtDNA COI gene. Significant population divergence was observed between individuals from Florida and those from Texas and Louisiana. In El Salvador, Central America, larvae of D. saccharalis were also collected and reared into adults. Larvae were collected in the rainy season and the dry season on different host plants including from sugarcane, corn, rice and sorghum. Individuals were similarly identified using morphological methods and a region of the mtDNA COI gene. There was modest host plant associated divergence of populations, and different natural enemies were collected on host plants in the rainy season than in the dry season. The genetic variability of D. saccharalis in a region could influence the choice and the success of different management practices.