The spongy moth Lymantria dispar L. is a major forest pest causing substantial economic damage in Holarctic region. Lymantria dispar multiple nucleopolyhedrovirus isolates (LdMNPVs) have demonstrated significant potential as biological control agents against this pest. In this study we evaluated the virulence of six LdMNPV isolates against five L. dispar populations (including subspecies dispar, asiatica, japonica) and L. umbrosa. Bioassay results revealed significant differences in susceptibility among the larvae, with the Krasnodar L. dispar population exhibiting notably higher susceptibility to LdMNPV infection than the Novosibirsk population, despite their shared host plant species. The non-local isolate LdMNPV-27/0 was more effective against Krasnodar larvae than the local LdMNPV-KR isolate. The LdMNPV-KG from Kyrgyzstan had minimal effect on larvae from Novosibirsk population but reduced survival in Krasnodar population. Our analyses indicated that virulence is predominantly determined by isolate-specific characteristics rather than the host population's origin or subspecies classification. Genomic analysis of novel LdMNPV sequences (LdMNPV-KR, -KG, and -BibJ) detected differences in genome sizes, gene composition, and specific mutations, including the absence of the enhancing factor-1 gene in the LdMNPV-BibJ isolate. Phylogenetic analysis confirmed the clustering of isolates into distinct clades reflecting their geographic origin and evolutionary history. Our findings suggest that LdMNPV's efficacy is primarily influenced by isolate-specific traits rather than by host origin or subspecies. The results contribute to a better understanding of genetic diversity of LdMNPV and offer valuable information for the development of more effective biological pest control strategies against L. dispar populations worldwide-irrespective of geographic or host subspecies variations-provided that the most effective isolate is selected for such an application.