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Publisher Copyright: © The Author(s) 2022. Published by Oxford University Press. Studying white matter connections with tractography is a promising approach to understand the development of different brain processes, such as proprioception. An emerging method is to use functional brain imaging to select the cortical seed points for tractography, which is considered to improve the functional relevance and validity of the studied connections. However, it is unknown whether different functional seeding methods affect the spatial and microstructural properties of the given white matter connection. Here, we compared functional magnetic resonance imaging, magnetoencephalography, and manual seeding of thalamocortical proprioceptive tracts for finger and ankle joints separately. We showed that all three seeding approaches resulted in robust thalamocortical tracts, even though there were significant differences in localization of the respective proprioceptive seed areas in the sensorimotor cortex, and in the microstructural properties of the obtained tracts. Our study shows that the selected functional or manual seeding approach might cause systematic biases to the studied thalamocortical tracts. This result may indicate that the obtained tracts represent different portions and features of the somatosensory system. Our findings highlight the challenges of studying proprioception in the developing brain and illustrate the need for using multimodal imaging to obtain a comprehensive view of the studied brain process.