Parcellation of the human thalamus based on cortical connectivity information inferred from non-invasive diffusion-weighted images identifies sub-regions that we have proposed correspond to nuclei. Here we test the functional and anatomical validity of this proposal by comparing data from diffusion tractography, cytoarchitecture and functional imaging. We acquired diffusion imaging data in eleven healthy subjects and performed probabilistic tractography from voxels within the thalamus. Cortical connectivity information was used to divide the thalamus into sub-regions with highest probability of connectivity to distinct cortical areas. The relative volumes of these connectivity-defined sub-regions correlate well with volumetric predictions based on a histological atlas. Previously reported centres of functional activation within the thalamus during motor or executive tasks co-localize within atlas regions showing high probabilities of connection to motor or prefrontal cortices, respectively. This work provides a powerful validation of quantitative grey matter segmentation using diffusion tractography in humans. Co-registering thalamic sub-regions from 11 healthy individuals characterizes inter-individual variation in segmentation and results in a population-based atlas of the human thalamus that can be used to assign likely anatomical labels to thalamic locations in standard brain space. This provides a tool for specific localization of functional activations or lesions to putative thalamic nuclei.