Abstract: Thirty eight isolates of Spodoptera frugiperda multiple nucleopolyhedrovirus(SfMNPV), collected from infected larvae on pastures, maize and sorghum plants in threedifferent geographical regions of Colombia, were subjected to molecular characterization andwere compared with a previously characterized Nicaraguan isolate (SfNIC). Restrictionendonuclease analysis (REN) showed two different patterns among Colombian isolates, oneprofile was particularly frequent (92%) and was named SfCOL. The physical map of SfCOL wasconstructed and the genome was estimated to be 133.9 Kb, with few differences in terms ofnumber and position of restriction sites between the genomes of SfNIC and SfCOL. The PstI-Kand PstI-M fragments were characteristic of SfCOL. These fragments were sequenced to revealthe presence of seven complete and two partial ORFs. This region was collinear with SfMNPVsf20 to sf27. However, two ORFs (4 and 5) had no homologies with SfMNPV ORFs, but werehomologous with Spodoptera exigua MNPV (se21 and se22/se23) and Spodoptera litura NPV(splt20 and splt21). Biological characterization was performed against two different colonies ofS. frugiperda, one originating from Colombia and one from Mexico. SfCOL OBs were twelvetimes more potent for the Colombian colony than SfNIC OBs. SfCOL and SfNIC showed aslower speed of kill (by ~50 h) in insects from the Colombian colony compared to the Mexicancolony, which was correlated with a higher production of OBs/larvae. SfCOL is a new strain ofSfMNPV that presents pathogenic characteristics that favor its development as the basis for abiopesticide product in Colombia.