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Deformed wing virus (DWV) is known as one of the main viruses that affect honeybees’ health all around the world. The virus has two widespread genotypes, DWV-A and DWV-B (VDV-1), transmitted mainly by <i>V. destructor</i> mites. In this study, we collected honeycombs with covered broods from 73 apiaries in eight Lithuanian regions and initially investigated the prevalence of <i>V. destructor</i> mites. Mites were collected from May to the end of July in 2021 from 124 hives. The prevalence of <i>V. destructor</i> infestations in beehives reached 30% and 63% in investigated apiaries. The presence of DWV-A and DWV-B pathogens in mites and broods was examined by RT-qPCR targeting the <i>CRPV-capsid</i> region. The molecular characterization of the virus in mite samples was based on sequence analysis of the RNA-dependent RNA polymerase (<i>RdRp</i>) region. In addition, leader polypeptide (<i>LP)</i>, structural protein (<i>Vp3)</i>, <i>Helicase</i>, and <i>RdRp</i> genes were used for phylogenetic characterization of dual infection. The prevalences of DWV-B in mites and broods were 56.5% and 31.5%, respectively, while DWV-A was detected in 12.9% of mite samples and 24.7% of brood samples. Some of the examined mite samples harboured dual virus infections. Our findings showed that bee colonies from the same apiary were not always infected by the same viruses. Some bee colonies were virus-free, while others were highly infected. Phylogenetic analysis of 21 sequences demonstrated the presence of highly variable DWV-B and DWV-A genotypes in Lithuania and possible recombinant variants of the virus. This study represents the first molecular characterization of mite-borne pathogens hosted by honeybees (<i>Apis mellifera</i>) in Lithuania.