In vivo tractography based on diffusion magnetic resonance imaging (dMRI) has opened new doors to study structure-function relationships in the human brain. Initially developed to map the trajectory of major white matter tracts, dMRI is used increasingly to infer long-range anatomical connections of the cortex. Because axonal projections originate and terminate in the gray matter but travel mainly through the deep white matter, the success of tractography hinges on the capacity to follow fibers across this transition. Here we demonstrate that the complex arrangement of white matter fibers residing just under the cortical sheet poses severe challenges for long-range tractography over roughly half of the brain. We investigate this issue by comparing dMRI from very-high-resolution ex vivo macaque brain specimens with histological analysis of the same tissue. Using probabilistic tracking from pure gray and white matter seeds, we found that ∼50% of the cortical surface was effectively inaccessible for long-range diffusion tracking because of dense white matter zones just beneath the infragranular layers of the cortex. Analysis of the corresponding myelin-stained sections revealed that these zones colocalized with dense and uniform sheets of axons running mostly parallel to the cortical surface, most often in sulcal regions but also in many gyral crowns. Tracer injection into the sulcal cortex demonstrated that at least some axonal fibers pass directly through these fiber systems. Current and future high-resolution dMRI studies of the human brain will need to develop methods to overcome the challenges posed by superficial white matter systems to determine long-range anatomical connections accurately. diffusion MRI | tractography | neuroanatomy | white matter | connectome T he primate cerebral cortex consists of dozens of areas distinguished by their cytoarchitectonic profiles (1), sensory maps (2), functional specialization (3), and spontaneous activity covariation (4). Attention within neuroscience has increasingly focused on understanding how connectivity among these regions underpins brain function in health and in disease (5). The macaque monkey (Macaca mulatta) has been a fruitful neuroscientific model because the functional organization of its brain is similar in many ways to that of the human (6-8). Tracer injections in the macaque have revealed that virtually all cortical areas give rise to long-range connections, many of which project to other cortical areas, potentially to form processing hierarchies (9). This understanding continues to shape views of functional organization in the human brain. However, studying long-range connections through tracer injections is time consuming, inefficient, and prone to sampling biases, because a given experiment can measure connectivity to only one or a small number of cortical sites. Moreover, although in many respects the macaque brain is a good approximation of the human brain, both species have undergone profound evolutionary changes since the time of their most recent...