Aims Nine commercial DNA extraction kits were evaluated for the isolation of DNA from 10-fold serial dilutions of Bacillus anthracis spores using quantitative real-time PCR (qPCR). The three kits determined by qPCR to yield the most sensitive and consistent detection (Epicenter MasterPure Gram Positive; MoBio PowerFood; ABI PrepSeq) were subsequently tested for their ability to isolate DNA from trace amounts of B.anthracis spores (approx. 6 center dot 5x101 and 1 center dot 3x102CFU in 25ml or 50g of food sample) spiked into complex food samples including apple juice, ham, whole milk and bagged salad and recovered with immunomagnetic separation (IMS). Methods and Results The MasterPure kit effectively and consistently isolated DNA from low amounts of B.anthracis spores captured from food samples. Detection was achieved from apple juice, ham, whole milk and bagged salad from as few as 65 +/- 14, 68 +/- 8, 66 +/- 4 and 52 +/- 16 CFU, respectively, and IMS samples were demonstrated to be free of PCR inhibitors. Conclusions Detection of B.anthracis spores isolated from food by IMS differs substantially between commercial DNA extraction kits; however, sensitive results can be obtained with the MasterPure Gram Positive kit. Significance and Impact of the Study The extraction protocol identified herein combined with IMS is novel for B.anthracis and allows detection of low levels of B.anthracis spores from contaminated food samples.