Brazil leads global lists of honeybee colony losses in South America as well as pesticide use, according to a web-based survey (http://www.nobeenofood.com/beealert). In association with that survey, Africanized honeybee (Apis mellifera ) samples were opportunistically collected when bee poisoning was apparently linked to pesticide use in crops. The objective was to determine concentrations of fipronil and neonicotinoids in live and dead honeybees, in areas where these compounds are widely used in agriculture. Pesticide residues in honeybees (54 live and 60 dead composite samples) were detected with mass spectrometry (UHPLC-MS/MS using QuEChERS methodology). Toxicological analyses in both matrices detected multiple contaminations with highest indices by fipronil with frequency of 55.3% and amplitude (0.7-23,539.7 ng/g), thiamethoxam 20.2% (0.6-13.6 ng/g), imidacloprid 3.5% (4.5-16.2 ng/g), nitenpyram 1.8% (3.8-7.4 ng/g), and thiacloprid 0.9% (1.6 ng/g). Neonicotinoids and fipronil residues had higher frequencies and amplitudes in honeybees collected near sugarcane plantations and orange orchards in northwest São Paulo state and other agro-industrial rural landscapes across the country dominated with fields of soybean, corn, and tropical fruit crops. These systemic pesticides were presumed to be primary mechanisms of honeybee colony losses in Brazil, according to a recently published 5-year survey by the same authors and reinforced by current analyses.