Honeybee health can be compromised not only by infectious and infesting diseases, but also by the acute or chronic action of certain pesticides. In recent years, there have been numerous reports of colony mortality by Italian beekeepers, but the investigations of these losses have been inconsistent, both in relation to the type of personnel involved (beekeepers, official veterinarians, members of the police force, etc.) and the procedures utilized. It was therefore deemed necessary to draw up national guidelines with the aim of standardizing sampling active ties. In this paper, we present the results of a survey carried out in Italy from 2015 to 2019, following these guidelines. Residues of 150 pesticides in 696 samples were analyzed by LC-MS/MS and GC-MS/MS. On average, 50% of the honeybee samples were positive for one or more pesticides with an average of 2 different pesticides per sample and a maximum of seven active ingredients, some of which had been banned in Europe or were not authorized in Italy. Insecticides were the most frequently detected, mainly belonging to the pyrethroid group (49%, above all tau-fluvalinate), followed by organophosphates (chlorpyrifos, 18%) and neonicotinoids (imidacloprid, 7%). This work provides further evidence of the possible relationship between complex pesticide exposure and honeybee mortality and/or depopulation of hives.