Dosidicus gigas is an economically important species distributed in the Eastern Pacific Ocean. Unraveling the genetic population structure of this species is crucial to ensure its fishery sustainability and management. Mitochondrial DNA sequences and nuclear neutral loci are useful to understand how historical and contemporary factors drive the genetic population structure of species. However, most studies investigating genetic structuring of D. gigas from its northern and southern populations rely on patterns identified using mitochondrial genes. The use of both types of DNA markers is especially relevant for marine species with high dispersal capabilities such as D. gigas. Here, we describe the genetic structure of D. gigas using partial sequences of the mitochondrial gene NADH dehydrogenase subunit 2 and nuclear microsatellite loci in populations of the northern hemisphere from the Costa Rica Thermal Dome and off Ecuador; and, of the southern hemisphere from the South Equatorial Current and off Peru. Statistical parsimony network and Bayesian analyses from mitochondrial sequences revealed three historical maternal lineages in both hemispheres, with high levels of genetic differentiation and signatures of population expansion during the late Pleistocene. Use of Discriminant Analysis of Principal Component (DAPC) with microsatellite loci of mature and immature individuals showed the presence of at least two contemporary genetic stocks homogeneously co-distributed in both northern and southern hemispheres, which can be explained by the biological characteristics of D. gigas and the variable oceanographic conditions of the Eastern Pacific Ocean. Overall, our findings indicate that cooperation between countries with intensive fishing will benefit the sustainability of D. gigas.