The effect of microstructural factors on hardness was investigated in normalized, tempered, aged and crept materials for Mod. 9Cr-1Mo steel, using nanoindentation and microhardness tests. Nanohardness and microhardness decreased during tempering, aging and creep exposure. Dislocation spacing, lath width, high angle boundary (block and packet boundary) spacing and inter-particle spacing increased during tempering, aging and creep exposure. A converted Vickers hardness was introduced to compare directly nanohardness and microhardness to Vickers hardness. The converted Vickers hardness increased with indent size in all the materials tested. Hardness at an indent size less than 1μm mainly consists of dislocations inside lath grains. Hardness at an indent size larger than 1μm originates from not only dislocation but precipitates and high angle boundaries such as block and packet boundaries. Comparing the converted Vickers hardness with lath width and high angle boundary spacing in normalized material with no precipitates, it was found that the lath boundary does not contribute to hardness. The difference in converted Vickers hardness between tempered and aged material was obviously large at the indent size, greater than inter-particle spacing. The decrease in hardness during aging is caused by increase in inter-particle spacing due to coarsening and coalescence of precipitates. On the other hand, not only changes in precipitates but also increase in high angle boundary spacing and dislocation spacing contribute to decrease in hardness during creep exposure.