The objective of this work was to create a fast and sensitive method of detecting Paenibacillus larvae from beehive debris based on PCR that does not require long-lasting cultivation steps. Various methods of extracting spores from beehive debris were compared: the original method of extraction of spores into toluene, and alternative spore extraction methods into Tween 80, into water, into isopropanol and into 95% ethanol. Isolation of DNA from various spore extractions was evaluated too. Best results were provided by isolation of DNA using the QIAamp DNA Mini Kit, without heat treatment. DNA of spores was detected by PCR from 0.25 g of beeswax debris, with the detected titer of 10(5) in 1g according to the cultivation tests.