The ventralis intermedius nucleus (Vim) is centrally placed in the dentato-thalamo-cortical pathway (DTCp) and is a key surgical target in the treatment of severe medically refractory tremor. It is not visible on conventional MRI sequences; consequently, stereotactic targeting currently relies on atlas-based coordinates. This fails to capture individual anatomical variability, which may lead to poor long-term clinical efficacy. Probabilistic tractography, combined with known anatomical connectivity, enables localisation of thalamic nuclei at an individual subject level. There are, however, a number of confounds associated with this technique that may influence results. Here we focused on an established method, using probabilistic tractography to reconstruct the DTCp, to identify the connectivity-defined Vim (cd-Vim) in vivo. Using 100 healthy individuals from the Human Connectome Project, our aim was to quantify cd-Vim variability across this population, measure the discrepancy with atlas-defined Vim (ad-Vim), and assess the influence of potential methodological confounds. We found no significant effect of any of the confounds. The mean cd-Vim coordinate was located within 1.88 mm (left) and 2.12 mm (right) of the average midpoint and 3.98 mm (left) and 5.41 mm (right) from the ad-Vim coordinates. cd-Vim location was more variable on the right, which reflects hemispheric asymmetries in the probabilistic DTC reconstructed. The method was reproducible, with no significant cd-Vim location differences in a separate test-retest cohort. The superior cerebellar peduncle was identified as a potential source of artificial variance. This work demonstrates significant individual anatomical variability of the cd-Vim that atlas-based coordinate targeting fails to capture. This variability was not related to any methodological confound tested. Lateralisation of cerebellar functions, such as speech, may contribute to the observed asymmetry. Tractography-based methods seem sensitive to individual anatomical variability that is missed by conventional neurosurgical targeting; these findings may form the basis for translational tools to improve efficacy and reduce side-effects of thalamic surgery for tremor.