BackgroundA lack of in utero imaging data hampers our understanding of the connections in the human fetal brain. Generalizing observations from postmortem subjects and premature newborns is inaccurate due to technical and biological differences.PurposeTo evaluate changes in fetal brain structural connectivity between 23 and 35 weeks postconceptional age using a spatiotemporal atlas of diffusion tensor imaging (DTI).Study TypeRetrospective.PopulationPublicly available diffusion atlases, based on 60 healthy women (age 18–45 years) with normal prenatal care, from 23 and 35 weeks of gestation.Field Strength/Sequence3.0 Tesla/DTI acquired with diffusion‐weighted echo planar imaging (EPI).AssessmentWe performed whole‐brain fiber tractography from DTI images. The cortical plate of each diffusion atlas was segmented and parcellated into 78 regions derived from the Edinburgh Neonatal Atlas (ENA33). Connectivity matrices were computed, representing normalized fiber connections between nodes. We examined the relationship between global efficiency (GE), local efficiency (LE), small‐worldness (SW), nodal efficiency (NE), and betweenness centrality (BC) with gestational age (GA) and with laterality.Statistical TestsLinear regression was used to analyze changes in GE, LE, NE, and BC throughout gestation, and to assess changes in laterality. The t‐tests were used to assess SW. P‐values were corrected using Holm‐Bonferroni method. A corrected P‐value <0.05 was considered statistically significant.ResultsNetwork analysis revealed a significant weekly increase in GE (5.83%/week, 95% CI 4.32–7.37), LE (5.43%/week, 95% CI 3.63–7.25), and presence of SW across GA. No significant hemisphere differences were found in GE (P = 0.971) or LE (P = 0.458). Increasing GA was significantly associated with increasing NE in 41 nodes, increasing BC in 3 nodes, and decreasing BC in 2 nodes.Data ConclusionExtensive network development and refinement occur in the second and third trimesters, marked by a rapid increase in global integration and local segregation.Level of Evidence3Technical EfficacyStage 2