Official control samples of wheat and rye flour were analyzed for the presence of Shiga-toxin producing Escherichia coli (STEC) in 25 g. The detection procedure was based on enrichment in buffered peptone water and Tryptone Bile X-Glucuronide agar, followed by multiplex real-time PCR with an internal positive control. Positive samples were sub-cultured for strain isolation by a two-step procedure. In the first step, ten colonies were picked from each plate, pooled, and analyzed by real-time PCR. In addition, a bacterial material from the part with heavy growth was analyzed. If the 1st isolation attempt failed, in a second step all colonies from molecular positive plates were picked, sub-cultured, pooled and analyzed. 39% of test samples were positive by real-time PCR. STEC was isolated from 17 test samples corresponding to 19%. Molecular detection is linked to the presence of quantifiable numbers of E. coli, whereas the grain species did not have an influence. There is a non-significant correlation with some mills indicating some technological or hygienic problems in those facilities. Increasing the number of subsamples did improve the detection rate. Our findings show the frequent presence of STEC in flour in Germany.