Abstract The complete genome of Lake Sinai virus 3 (LSV3) was sequenced by the Ion Torrent next-generation sequencing (NGS) technology from an archive sample of honey bees collected in 2010. This strain M92/2010 is the first complete genome sequence of LSV lineage 3. From October 2016 to December 2017, 56 honey bee samples from 32 different locations and 41 bumble bee samples from five different locations were collected. These samples were tested using a specific reverse transcriptase-polymerase chain reaction (RT-PCR) method; 75.92% of honey bee samples and 17.07% of bumble bee samples were LSV-positive with the RT-PCR method. Phylogenetic comparison of 557-base pair-long RNA-dependent RNA polymerase (RdRp) genome region of selected 23 positive samples of honey bees and three positive bumble bee samples identified three different LSV lineages: LSV1, LSV2, and LSV3. The LSV3 lineage was confirmed for the first time in Slovenia in 2010, and the same strain was later detected in several locations within the country. The LSV strains detected in bumble bees are from 98.6 to 99.4% identical to LSV strains detected among honey bees in the same territory.