The microhardness of poly(ethylene naphthalene-2,6-dicarboxylate) (PEN), with a detailed characterized nanostructure has been investigated. PEN samples were crystallized from the glassy state at atmospheric pressure and from the melt at high pressure and were characterized using small-angle X-ray scattering (SAXS), wide-angle X-ray scattering (WAXS) and differential scanning calorimetry (DSC). Results show that the degree of crystallinity derived from WAXS, for both atmospheric and high-pressure crystallized PEN, is smaller than that obtained by density and calorimetry. For high-pressure crystallized samples, both, crystallinity and microhardness values are larger than those found for the material crystallized under atmospheric pressure. In the latter case, the hardness values depend on the volume fraction of lamellar stacks within spherulites X-L that depends on the crystallization temperature T-c. For T-c < 200 degreesC, X-L is found to be less than 50%. Thus, for T-c < 200 degreesC a linear relationship between H and Tc is observed provided a sufficiently long crystallization time is used. Results are discussed in terms of the rigid amorphous fraction that appears as a consequence of the molecular mobility restrictions due to the crystal stacks.