Author Summary With the advent of ultrahigh-resolution functional MRI (fMRI), increased noninvasive accessibility of the human cortical laminae has enabled more advanced study of the human cortex. One potential methodological approach to studying cortical laminae is through multilayer connectomics, whereby the human cortex is represented by several interconnected networks. We applied a multilayer graph theory to examine functional connectivity across different cortical depths in humans, using 7-T fMRI. We utilized global and local graph theory features that quantitatively characterize network attributes including network composition, nodal centrality, path-based measures, and hub segregation. We detected global and local functional differences between cortical depths that were augmented using multilayer connectomics compared with the layer-by-layer versions. Thus, multilayer connectomics could provide a methodological framework for studies on how the human cortex functions. Abstract In graph theory, “multilayer networks” represent systems involving several interconnected topological levels. One example in neuroscience is the stratification of connections between different cortical depths or “laminae,” which is becoming noninvasively accessible in humans using ultrahigh-resolution functional MRI (fMRI). Here, we applied multilayer graph theory to examine functional connectivity across different cortical depths in humans, using 7-T fMRI (1-mm 3 voxels; 30 participants). Blood oxygenation level dependent (BOLD) signals were derived from five depths between the white matter and pial surface. We compared networks where the interregional connections were limited to a single cortical depth only (“layer-by-layer matrices”) with those considering all possible connections between areas and cortical depths (“multilayer matrix”). We utilized global and local graph theory features that quantitatively characterize network attributes including network composition, nodal centrality, path-based measures, and hub segregation. Detecting functional differences between cortical depths was improved using multilayer connectomics compared with the layer-by-layer versions. Superficial depths of the cortex dominated information transfer, and deeper depths drove clustering. These differences were largest in frontotemporal and limbic regions. fMRI functional connectivity across different cortical depths may contain neurophysiologically relevant information; thus, multilayer connectomics could provide a methodological framework for studies on how information flows across this stratification.