Subcortical white matter (SWM) is a thin layer of white matter (WM) residing just below the cortical sheet. Its structure, metabolism and function differ substantially from deep WM. Importantly, SWM incorporates short association fibers, the intra-hemispheric connections between adjacent gyri, referred to as U-fibers. Despite their importance for cortico-cortical connectivity little is known about the density and distribution of U-fiber in humans¹ mainly due to the lack of appropriate imaging methods. In humans invasive tracer studies are not applicable and DTI-based tractography does not yield satisfactory results in SWM². Several SWM contrasts has been demonstrated with structural MRI ex-vivo at 1.5T³ and 7T⁴ and in-vivo at 7T⁵, but no study has fully explored the potential of high-resolution ultra-high field structural SWM mapping in-vivo yet. Thus, we investigate four contrasts in SWM ((i) quantitative transverse and (ii) effective transverse relaxation rates (qR₂ and qR₂*), (iii) longitudinal relaxation rate (qR₁) and (iv) quantitative susceptibility maps (QSM)) using high-resolution in-vivo MRI at 7T. We show that SWM qR₂*, qR₂ and QSM exhibit strong contrast to both grey matter (GM) and deep WM. Iron and iron reach cells were identified as the intrinsic contrast agents by comparing the in-vivo data with ex-vivo MR imaging and quantitative iron maps acquired with proton induced X-ray emission (PIXE).MR measurements were performed on a whole body 7 T Siemens MRI scanner. qR₂* and qR₁ maps with an isotropic resolution of 0.5 mm were sourced from a recently published repository and analysed⁶. qR₁ maps were co-registered in the coordinate space of the qR₂* maps using 0.6 mm resolution R₂* and T₁-weighted images, acquired in a separate session. WM and GM regions were segmented based on the co-registered qR₁ maps⁷. For two participants QSM were reconstructed with the HEIDI algorithm⁸ employing phase images of an additional multi-echo gradient-echo (GRE) acquisition (0.5 mm isotropic). For one volunteer an additional multi-echo spin-echo data set (0.5 in plane resolution, 1 mm slices) has been acquired and used for calculating transverse relaxation rate (qR₂) maps. To better understand the in-vivo maps, post mortem tissue samples were additionally investigated with T₂*-weighted imaging (FLASH, 80 μm isotropic resolution) and qR1 mapping (MP2RAGE, 200 μm resolution, occipital pole, 92 y, f, postmortem time before fixation 22 hours) as well as quantitative iron mapping with PIXE (primary motor and sensory cortex, 82 y, f, postmortem time before fixation 8 h)⁹.High-resolution in-vivo 7T MRI revealed a thin (0.5-2 mm) hyperintense layer below the GM-WM border on qR₂, qR₂*, and QSM maps (see Figs.1 and 2), indicating SWM. qR₂ (ΔR₂=8±3 s^-1), qR₂* (ΔR₂*=15±5 s^-1), and magnetic susceptibility (Δχ=25±5 ppb) values in this layer were significantly higher than in deep WM. Interestingly, the increase in qR₂, qR₂*, QSM was found to be more prominent in sulcal fundi and walls as compared to the gyral crowns. Similar findings were observed on the ex-vivo tissue sample, where SWM again exhibited as a thin contrast-rich layer with decreased signal intensities on T2*-weighted images and hyperintense in qR₁ (ΔR₁=0.4±0.1 10^-3 s-1) maps (see Fig. 3), below the GM-WM border. Contrast between SWM and deep WM was found to be stronger, than the intra-cortical contrast in both T₂*-weighted and qR₁ images. Iron quantification yielded increased iron levels within a 1 mm thick SWM layer below the GM-WM boarder, whereas iron content has been determined to be 355 ± 30 μg, 255 ± 50 μg, 92 ± 30 μg per g dry brain tissue in SWM, GM, and WM, respectively.Utilizing the superior resolution provided by 7 T MRI we were able to image SWM in in-vivo and in post-mortem brain tissue samples at the WM-GM interface. A clear-cut discrimination of SWM from the adjacent brain regions was obtained based on higher qR₂*, qR₂ and susceptibility in in-vivo tissue and higher qR₂* and qR₁ in ex-vivo tissue as compared to both GM and deep WM. We found the SWM contrast to be even higher than the GM-WM contrast as well as intra-cortical grey matter contrast. The higher R₂, R₂*, and susceptibility values indicate iron complexes and iron reach cells in SWM as dominating biophysical contrast source and is further supported by the elevated iron concentrations in SWM measured with PIXE. These new findings are of paramount importance and may pave the way for future in-vivo segmentation strategies for this crucial brain region as well as potential U-fiber density mapping in humans.