The presence of mycotoxins, in particular aflatoxin B1, can cause significant health problems as well as severe societal economic losses, and is therefore regulated with respect to maximum acceptable concentration in various feed- and foodstuffs. International regulatory authorities have begun to recognize the importance of representative sampling, but sampling guidelines are only partly in compliance with the Theory of Sampling (TOS). In particular, practical guidance regarding sampling, including correct design and operation of sampling devices, including explanation on how to develop sufficient sampling protocols are lacking in current guidelines. These are critical practicalities of main importance, especially when dealing with trace concentrations and/or concentrations that are irregularly distributed—as is the case for mycotoxins. Furthermore, heterogeneity characterization, which is a necessary requirement to be able to develop valid sampling protocols or validation assessments of existing sampling operations, is currently not mentioned in the existing guidelines. The present paper focuses on heterogeneity characterization with respect to sampling of mycotoxins for 1-D and 3-D feed lots (a full analysis of all critical practicalities in sampling mycotoxins is published elsewhere). Structural guidelines for correctly designing experimental heterogeneity characterizations are presented, allowing evaluation of sampling representativeness and determination of optimal number of increments per composite sample.