Fifteen lots of wheat were sampled to characterise the total variance and distribution among sample test results associated with measuring deoxynivalenol (DON) in bulk wheat lots. An unbalanced nested experimental design based on past research was used to determine contributions to the total variance from sampling, sample preparation, and analysis. The wheat lots used in the study contained average DON concentrations that ranged from 0.17 to 24.5 mg/kg. Sampling was determined to be the largest contributor to the total variance of measuring DON at low mg/kg concentrations, which are relevant to existing maximum levels. With the experimental design parameters of 1 kg laboratory samples, sub-division of whole and ground grain using rotary sample division, sample comminution using a commercial-grade coffee grinder, extraction of 100 g test portions, and making one measurement of DON in the test portion by gas chromatography-mass spectrometry, the total variance of DON measurement at 2 mg/kg was 0.046 mg2/kg2 (coefficient of variation=10.7%). At this concentration, sampling contributed 67% to the total variance, followed by sample preparation (18%) and analysis (15%). The DON distribution among sample test results was accurately described by the normal distribution. The mathematical model of variance was used with the normal distribution of DON measurement results to construct operating characteristics curves to model the likelihood of mischaracterising a wheat lot as (non) compliant with a certain decision limit. With realistic laboratory sample and test portion sizes, as well as a practicable decision limit of 1.5 mg/kg, the estimated probability of mischaracterising a wheat lot containing 2 mg/kg DON as less than this concentration was reduced to 1%.