Worldwide, the fall armyworm Spodoptera frugiperda (J.E. Smith) is considered the most important pest of maize crop. Nucleopolyhedrovirus of Baculoviridae family have been successfully used in several countries for its control. However, in Colombia there are not any biopesticide registered for the control of this pest. In the present work, native nucleopolyhedrovirus were isolated from the departments of Tolima, Meta and Cordoba, by collecting larvae in different crops. Samples were analyzed by microscopy in order to find viral particles and the isolates were characterized physically and biologically. A total of 2,140 larvae of S. frugiperda were collected in the three sampled zones. However, larvae with viral infection symptoms in the field were found only in one place (Cerete, Cordoba). The 2% of the samples exhibited typical symptoms of infection and 3 isolates were identified and codified as NPV003 (Cordoba), NPV009 (Tolima) and NPV011 (Meta). Isolates showed a particle size between 1.93 μm and 2.14 μm. The mean lethal concentrations (LC 50 ) over neonate larvae ranged between 2.2 x 105 CI/mL and 7.0 x 105 CI/mL and there were not statistical differences among isolates, suggesting that all have the same pathogenicity on neonate larvae of the insect. Currently, these native viruses represent the base for developing a new biological alternative for the control of this pest, being necessary to being the studies for their formulation