► Seven neonicotinoids were simultaneously determined in beeswax for the first time. ► The usefulness of fused-core columns to separate neonicotinoids was demonstrated. ► The sample clean-up was performed onto diatomaceous based cartridges. ► Matrix (beeswax) had an influence (ion suppression) onto the ESI-MS signals. ► Residues of thiamethoxam, acetamiprid and imidacloprid were found in 11 samples. A new method has been developed to measure seven neonicotinoid insecticides (acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram, thiacloprid and thiamethoxam) in beeswax using liquid chromatography (LC) coupled to electrospray ionization mass spectrometry (ESI-MS) detection. Beeswax was melted and diluted in an n-hexane/isopropanol (8:2, v/v) mixture. After this, liquid extraction with water was performed followed by a clean-up on diatomaceous material based cartridges. The compounds were eluted with acetone, and the resulting solution was evaporated until dry and reconstituted with a mixture of water and acetonitrile 50:50 (v/v). The separation of all compounds was achieved in less than 15min using a C18 reverse-phase fused-core column (Kinetex® C18, 150mm×4.6mm i.d.) and a mobile phase composed of a mixture of 0.1% formic acid in water and acetonitrile in gradient elution mode at 0.5mL/min. This method was fully validated in terms of selectivity, linearity, precision and recovery. Low limits of detection and quantification could be achieved for all analytes ranging from 0.4 to 2.3μg/kg, and from 1.5 to 7.0μg/kg, respectively. Finally, the proposed method was applied to an analysis of neonicotinoid residues in beeswax samples from apiaries located close to fruit orchards.