The aim of this study was to investigate the distribution of fumonisin B1 in various corn milling fractions processed by an industrial plant. Corn kernels and six derived milling fractions (germ, bran, large and small grits, animal feed flour, and flour) were sampled. In addition, in order to evaluate the effect of cooking, samples of polenta were prepared starting from naturally contaminated flour obtained from the industrial processing cycle. The industrial plant worked continuously at a rate of 60 tons per day. Two sublots of 5 tons each were investigated with samples of derived products taken at regular time intervals. Due to a similar heterogeneous distribution of fumonisin B1 with other mycotoxins, such as aflatoxins, the sampling scheme was derived from the European Directive 98/53 for aflatoxins. Both lots of kernels showed fumonisin contamination at 4.54 and 5.09 mg/kg, respectively. Germ, bran, and animal feed flour showed contamination levels, namely 8.92 mg/kg (lot 1) and 9.56 mg/kg (lot 2), 7.08 mg/kg (lot 1) and 8.08 mg/kg (lot 2), and 9.36 mg/kg (lot 1) and 6.86 mg/kg (lot 2) higher than large and small grits and flour (0.39 mg/kg [lot 1] and 0.42 mg/kg [lot 2], 0.60 mg/kg [lot 1] and 1.01 mg/kg [lot 2], and 0.40 mg/kg [lot 1] and 0.45 mg/kg [lot 2], respectively). These results seem to account both for the industrial yields of the derived products and the distribution of fumonisin contamination in a kernel. The cooking of polenta in a domestic pressure cooker did not affect fumonisin contamination because the mycotoxin concentrations were similar to those of the starting flour (0.40 and 0.45 mg/kg).