Background: White matter is responsible for inter-neuronal connections throughout the brain that are a driving force in cognitive development. Diffusion tensor imaging (DTI) fiber tractography has been used to evaluate white matter development in the fetal brain; however, longitudinal studies of DTI fiber tractography to assess white matter development in the third trimester are lacking. Objective: To characterize in utero longitudinal changes in the fetal brain DTI fiber tracts of normal third-trimester fetuses. Materials and methods: For this single-center prospective longitudinal observational pilot study, we recruited 28 pregnant females with normal third-trimester pregnancies who had routine prenatal ultrasound. MRI of the in utero fetal brain was performed with a Siemens 1.5-tesla (T) Espree scanner at 31 weeks, 33 weeks and 36 weeks of gestation, with 14 DTI tractography parameters quantified in 7 brain regions using DTI-studio version 2.4 (Johns Hopkins University, Baltimore, MD; n=98 measurements). We used multilevel mixed models to examine the relationship between longitudinal changes in DTI measurements and between 98 DTI measurements at 31 weeks and 4 routine fetal brain anatomical biometrics (n=392 assessments). Results: We observed statistically significant decreases in radial diffusivity and apparent diffusion coefficient in 13 of 14 brain regions from 31 weeks to 36 weeks of gestation (P<0.001 for all regions except the genu of the corpus callosum). Significant decreases in radial diffusivity from weeks 33 to 36 and weeks 31 to 36 were seen in the corticospinal tracts, centrum semiovale, posterior limb of the internal capsule, and crus cerebri (P<0.001 for all). When considering all possible combinations of DTI fiber tract measurements and the routine morphological fetal brain biometrics, only 6% (24/392) had a significant association (P<0.05), indicating relative independence of the DTI fiber tract measurements from anatomical biometrics. Conclusion: In utero longitudinal changes in fetal brain DTI fiber tractography are quantifiable in normal third-trimester fetuses and are largely independent of morphological brain changes.