Objective To explore quantitatively and qualitatively how the number of gradient directions (NGD) and spatial resolution (SR) affect diffusion tensor imaging (DTI) tractography in patients planned for brain tumor surgery, using routine clinical magnetic resonance imaging protocols. Methods Of 67 patients with intracerebral lesions who had 2 different DTI scans, 3 DTI series were reconstructed to compare the effects of NGD and SR. Tractographies for 4 clinically relevant tracts (corticospinal tract, superior longitudinal fasciculus, optic radiation, and inferior fronto-occipital fasciculus) were constructed with a probabilistic tracking algorithm and automated region of interest placement and compared for 3 quantitative measurements: tract volume, median fiber density, and mean fractional anisotropy, using linear mixed-effects models. The mean tractography volume and intersubject reliability were visually compared across scanning protocols, to assess the clinical relevance of the quantitative differences. Results Both NGD and SR significantly influenced tract volume, median fiber density, and mean fractional anisotropy, but not to the same extent. In particular, higher NGD increased tract volume and median fiber density. More importantly, these effects further increased when tracts were affected by disease. The effects were tract specific, but not dependent on threshold. The superior longitudinal fasciculus and inferior fronto-occipital fasciculus showed the most significant differences. Qualitative assessment showed larger tract volumes given a fixed confidence level, and better intersubject reliability for the higher NGD protocol. SR in the range we considered seemed less relevant than NGD. Conclusions This study indicates that, under time constraints of clinical imaging, a higher number of diffusion gradients is more important than spatial resolution for superior DTI probabilistic tractography in patients undergoing brain tumor surgery.