Diffusion weighted imaging (DWI) and tractography allow the non-invasive study of anatomical brain connectivity. However, a gold standard for validating tractography of complex connections is lacking. Using the porcine brain as a highly gyrated brain model, we quantitatively and qualitatively assessed the anatomical validity and reproducibility of in vitro multi-fiber probabilistic tractography against two invasive tracers: the histochemically detectable biotinylated dextran amine and manganese enhanced magnetic resonance imaging. Post mortem DWI was used to ensure that most of the sources known to degrade the anatomical accuracy of in vivo DWI did not influence the tracking results. We demonstrate that probabilistic tractography reliably detected specific pathways. Moreover, the applied model allowed identification of the limitations that are likely to appear in many of the current tractography methods. Nevertheless, we conclude that DWI tractography can be a precise tool in studying anatomical brain connectivity.