Spodoptera frugiperda J. E. Smith (Lepidoptera: Noctuidae) is a moth that has genetically diverged in two strains named “corn” and “rice.” This study was focused on carrying out a molecular characterization of the species with Amplified Fragment Length Polymorphism (AFLP) using larvae collected in the departments of Córdoba (North), Meta (East), and Valle del Cauca (West) in corn, cotton, and rice fields, to improve the information of the species obtained in 2008. In this work, 134 DNA extractions were made from each larva head and 45 samples were amplified with three combinations of AFLP primers that produced 101 loci. The AFLP were useful to determine that populations of S. frugiperda of Colombia have a high genetic variability, but they do not differentiate the strains as other authors reported. The analysis of molecular variance (AMOVA) showed that populations of S. frugiperda from the three departments were not genetically differentiated. The UPGMA dendrogram obtained also showed that there is no a genetic differentiation of the genotyped individuals based on the geographical location or the crop sampled. Our results coincide with a previous report obtained with the same species of Colombia based on a sequence of the mitochondrial gene cytochrome oxidase I (COI).