Hyperostosis frontalis interna (HFI) represents irregular thickening of the endocranial surface of the frontal bone, mostly seen in postmenopausal females. The microarchitecture of this condition is poorly studied. The aim of this cross-sectional autopsy study was to investigate and compare microarchitectural structure of the frontal bone affected with HFI in both sexes and to test whether HFI severity could be distinguished at the microarchitectural level. The sample was taken from human donor cadavers, 19 males (61 ± 15 years old) and 17 females (75 ± 15 years old). After classification of HFI severity (type A, B, C or D), samples of the frontal bone were taken and scanned using micro-computed tomography. Bone volume fraction was higher and total porosity lower only in the outer table of males with HFI, compared to females with HFI. Mean total sample thickness differed only between males with HFI type A and D. Bone microarchitecture between males and females with corresponding HFI types (e.g., male with type A versus female with type A) differed only in HFI type C regarding the fractal dimension of diploe. The degree of anisotropy differed between HFI subtypes in males, but the post hoc analysis revealed no significant differences between individual groups. Other microarchitectural parameters did not differ among males with different HFI subtypes, as well in females, in any part of the frontal bone. There is no difference in microarchitectural structure of the frontal bone between males and females with HFI, in general aspect and within corresponding HFI subtypes. HFI severity could not be distinguished at the microarchitectural level, neither in males nor in females.