Aim: To develop a real-time PCR-based strategy for the detection of Paenibacillus larvae vegetative cells and spores to improve the diagnosis and the screening of American foulbrood (AFB), the most harmful pathology of honeybee brood. Methods and Results: A real-time PCR that allowed selective identification and quantification of P. larvae 16S rRNA sequence was developed. Using standard samples quantified by flow cytometry, detection limits of 37·5 vegetative cells ml-1 and 10 spores ml-1 were determined. Compared to spread plate method, this real-time PCR-based strategy allowed, in only 2 h, the detection of P. larvae in contaminated honeys. No false-positive results were obtained. Moreover, its detection limit was 100 times lower than that of the culture method (2 vs 200 spores g-1 of honey). Conclusion: A rapid, selective, with low detection limit, sensitive and specific method to detect and quantify vegetative cells and spores of P. larvae is now available. Significance and Impact of Study: In addition to honey samples, this real-time PCR-based strategy may be also applied to confirm AFB diagnosis in honeybee brood and to screen other apiary supplies and products (bees, pollen, wax), thus broadening the control of AFB spreading.