Premature infants exhibit widespread insults and delays in white matter maturation that can be sensitively detected early using diffusion tensor imaging. Diffusion tensor tractography facilitates in vivo visualization of white matter tracts and has the potential to be more sensitive than simpler two-dimensional DTI-based measures. However, the reliability and reproducibility of performing tractography for major white matter tracts in preterm infants is not known. The main objective of our study was to develop highly reliable and repeatable methods for ten white matter tracts in extremely low birth weight infants (birth weight ≤ 1000 g) at term-equivalent age. To demonstrate clinical utility, we also compared fiber microstructural and macrostructural parameters between preterm and healthy term controls. Twenty-nine ELBW infants and a control group of 15 healthy term newborns were studied. A team of researchers experienced in neuroanatomy/neuroimaging established the manual segmentation protocol based on a priori anatomical knowledge and an extensive training period to identify sources of variability. Intra- and inter-rater reliability and repeatability was tested using intra-class correlation coefficient, within-subject standard deviation (SD), repeatability, and Dice similarity index. Our results support our primary goal of developing highly reliable and reproducible comprehensive methods for manual segmentation of 10 white matter tracts in ELBW infants. The within-subject SD was within 1-2% and repeatability within 3-7% of the mean values for all 10 tracts. The intra-rater Dice index was excellent with a range of 0.97 to 0.99, and as expected, the inter-rater Dice index was lower (range: 0.80 to 0.91), but still within a very good reliability range. ELBW infants exhibited fewer fiber numbers and/or abnormal microstructure in a majority of the ten quantified tracts, consistent with injury/delayed development. This protocol could serve as a valuable tool for prompt evaluation of the impact of neuroprotective therapies and as a prognostic biomarker for neurodevelopmental impairments.