The European honeybee (Apis mellifera) is a key pollinator and has in the last decades suffered significant population decline. A combination of factors, including decrease in genetic diversity and introduction of Varroa mites, have been suggested to be responsible for these losses, but no definitive cause has yet been appointed. In Europe not only have wild colonies been severely affected, but managed hives have had a massive decline in numbers. To test the hypothesis that honeybees' genetic diversity has decreased in the recent past, we used reduced representation genome sequencing of 40 historical honeybee specimens collected in Natural History collections across Europe and compared them to genomic data from 40 individuals from extant populations (collected post 2006). Our results are consistent with the existence of five evolutionary lineages as previously described, and show a decrease in genetic diversity between historical and extant individuals of the same lineage, as well as high levels of admixture in historical specimens. Our data confirm that a loss of genetic diversity has occurred during the last century, potentially increasing honeybees' vulnerability to contemporary ecological and anthropogenic stressors.