Four genotypes named SP2A, SP2B, SP2C and SP2D were obtained in vivo by infecting S. exigua larvae with limiting dilutions of the Spanish field isolate Spodoptera exigua Nucleopolyhedrovirus (Se-SP2) of SeMNPV. The cloning of variants SP2A, SP2B and SP2C took 1, 6, and 3 passages, respectively, before the DNA profiles showed all bands in equimolar concentrations, and they remained constant for at least six further passages indicating the stability of their genotypes. The SP2D variant isolation took over ten passages and it was genetically less stable. Physical maps of their genomes were constructed for the restriction enzymes BamHI, BglII, PstI, and XbaI. The region between 8-10 m.u. was highly variable and characteristic of each cloned genotype and, hence, can be used as RFLP markers for all four genotypic variants. This region, included in the PstI-MB fragment, was cloned and sequenced showing that all the Se-SP2 variants contained a homologous region (hr) with a variable number of 98 bp sequences tandemly repeated, which were used to distinguish genotypic variants from each other. The biological activity of the genotypic variants SP2A, SP2B, and SP2C when compared in terms of LD50 and LT50, were not significantly different. However, the SP2D genotypic variant was found to be significantly less infective (higher LD50). The emergence of new genotypes in the Se-SP2 field populations is discussed.