Spodoptera frugiperda (Smith) is a polyphagous insect of major economic impact in the Western hemisphere, and it has genetically differentiated into two strains (i.e., corn and rice) that are morphologically identical at the level of larvae and adult but differs in their wing shape and in their genetics. In 2008, corn and rice strains of this pest were identified from Tolima department in Colombia. This work continues the molecular identification of these two strains in three other regions of Colombia and three other crops (grass, sugarcane, and sweet sorghum). Our results showed that both strains are associated to the same hosts as reported in 2008. The corn strain was more abundant in corn, cotton, sorghum, sugarcane, and sweet sorghum, whereas the rice strain was more abundant in grass and rice. This host plant association reflects a population genetic differentiation in Colombia, with significant genetic differentiation between subpopulations (PhiPT) values for both markers—cytochrome oxidase I and FR (For Rice) fragment repeat—for all crops. The UPGMA (Unweighted Pair Group Method with Arithmetic Mean) dendrograms obtained with Nei genetic distances separated most of rice strain populations from the other crops, suggesting that it is genetically differentiated from the corn strain. Our results demonstrate the importance of differentiating these two strains in nature, as these two populations significantly differ in their tolerance to both insecticides and Bacillus thuringiensis endotoxins under laboratory conditions and thus transference of genes that confer resistance to any control is low.