Real-time polymerase chain reaction (PCR)-based assays for detection of cashew (Anacardium occidentale) and macadamia nut (Macadamia intergrifolia) traces in food products are described here. The real time PCR technique proposed herein were developed based on the design of macadamia and cashew-specific primers from the ITS region and a TaqMan fluorescent probe. The methods were positive for cashew and macadamia respectively, and negative for all other heterologous plant and animal species tested. Using a series of model samples with defined levels of raw and heat-treated cashew and macadamia nut, respectively, within a range of concentrations of 0.1–100 000 mg kg⁻¹, practical detection limits of 0.1 mg kg⁻¹ for cashew and macadamia nut were estimated. Practical applicability of the PCR methods was tested by the analysis of a total of 214 commercial foodstuffs. These PCR methods, are useful for highly selective, and sensitive detection of traces of macadamia and cashew nuts in commercial food products and is therefore proposed as a ready-to-use analytical tool to trace these target allergens in foods.