Spodoptera frugiperda (Smith) is a polifagous insect of major economic impact in the western hemisphere and exhibits two strains (i.e., corn and rice) that are morphologically identical but differ in ecology, genetics and physiology. In this work we identified these strains and their respective hybrids by using a PCR-RFLP of the COI gene and PCR of the tandem region FR. Moreover, we performed a population structure analysis by using 253 larvae from Tolima, a region where S. frugiperda is a pest on corn, rice, sorghum and cotton. Corn strain was found on 42% in corn, 34% in cotton, 19% in sorghum and 0.04 % in rice and rice strain on 35% in corn, 0.06% in cotton, 0.06% in sorghum and 53% in rice, demonstrating that corn strain specificity is superior to rice strain. Hybrids between these strains were more abundant in corn. The distributions on their host plants reflect a population genetic differentiation in S. frugiperda with values of PhiPT (COI) = 0.31, P < 0.0001, PhiPT (FR) = 0.17, P < 0.0001 for all crops and PhiPT (COI) = 0.42, P < 0.01, PhiPT (FR) = 0.13, P < 0.01 for the sixteen sampled farms. The dendrograms showed two clusters representing both strains. The results obtained in this study suggest that the management of this insect must differ on each host plant, given the specialization that both strains present, particularly in corn and rice.