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The application of a method combining x‐ray microscopy and diffraction analysis to the study of the substructure of nickel and nickel alloys disclosed the coexistence of three orders of magnitude of substructural entities. These three orders are structurally interrelated and distinguished by different ranges of size, disorientation angle, and lattice misalignment. The statistical analysis of the data disclosed that for the two largest orders of substructural entities increasing amounts of room temperature deformation cause (1) a decrease in the mean size and a decrease in the spread of the size distribution, (2) an increase in the mean disorientation angle between substructural entities of the same order and an increase in the spread of the distribution of angles, and (3) an increase in the mean lattice misalignment and spread in the distribution of misalignment. Progressive room temperature deformation causes the low‐angle boundaries to become increasingly unsymmetrical and greatly increases the number of the smallest substructural entities. The rise of flow stress as a function of room temperature deformation was correlated to the systematic modification of substructure characteristics.