Diatraea saccharalis (Fabricius) is one of the major Lepidopteran pests throughout the Western Hemisphere. In Argentina, it causes significant losses mainly in sugarcane and corn. Previous studies determined the existence of reproductive incompatibilities between populations from Buenos Aires and Tucumán from different host plants; however, the genetic base of this incompatibility is still unknown. The aim of this study was to determine the existence of genetic structure of D. saccharalis populations collected from different hosts and regions in Argentina, comparing with those from Brazil, through COI and NGS. The COI analyzes showed a haplotypic diversity of 0.8 and a Nucleotide diversity of 0.0058. AMOVA revealed a greater variation within populations than among them. The 17 haplotypes detected were linked in a single parsimony network that did not reveal clusters clearly influenced by geographic locations or the host plants from which populations were collected. A total of 4.549 SNPs were obtained through NGS. Out of the 2,349 outlier loci, 84 showed similarities with known proteins previously characterized. The coefficient of inbreeding showed that the mating among the populations, although it is being random, presents the same degree of selection. The FST values showed a high variation among Argentina and Brazil populations; however, did not show a clearly defined trend of the influence of the host and/or the distance. In the DAPC, three different groups were formed, a group that includes the samplings from Brazil and two groups from Argentina. Results indicated the existence of genetic divergence in D. saccharalis populations.