To provide a scientific sound assessment of the prevalence and levels of mycotoxins in U.S. distillers' dried grains with solubles (DDGS), we measured mainly aflatoxins, deoxynivalenol, fumonisins, T-2 toxin, and zearalenone in 235 DDGS samples collected from 20 ethanol plants in the midwestern United States and 23 export shipping containers from 2006 to 2008 using state-of-the-art analytical methodologies. The results suggested that (1) none of the samples contained aflatoxins or deoxynivalenol levels higher than the U.S. Food and Drug Administration (FDA) guidelines for use in animal feed; (2) no more than 10% of the samples contained fumonisin levels higher than the recommendation for feeding equids and rabbits, and the rest of the samples contained fumonisins lower than FDA guidelines for use in animal feed; (3) none of the samples contained T-2 toxins higher than the detection limit, and no FDA guidance levels are available for T-2 toxins; (4) most samples contained zearalenone levels lower than the detection limit, and no FDA guidance levels are available for zearalenone; and (5) the containers used for export shipping of DDGS did not seem to contribute to mycotoxin production. This study was based on representative DDGS samples from the U.S. ethanol industry, and the data were collected using reference methods. This study provided a comprehensive and scientifically sound assessment of the occurrence and levels of mycotoxins in DDGS from the U.S. ethanol industry.