The nature of how human γδ T cells are normally generated is not clear. We have used an RT-PCR assay and DNA sequencing to identify and compare δ-encoded TCRs (TCRDs) that are generated de novo in the fetal gut, liver, and thymus and to determine when, where, and how the TCRD repertoire is established during normal embryonic development. Rearranged TCRDV genes are first expressed outside of the thymus in the liver and primitive gut between 6 and 9 wk gestation. Although DV1Rs and/or DV2Rs predominated, differences in the pattern of TCRDV gene rearrangement and transcription in each tissue during ontogeny were identified. Specific, DV2-encoded TCRs are highly conserved throughout ontogeny in the tissues from the same and between genetically distinct donors. Although the thymic and intestinal γδ T cell repertoires partially overlap early in development, they diverge and become nonoverlapping during the second trimester, and the generation of the intestinal γδ T cell repertoire is characterized by differences in the processing of DV1Rs and DV2Rs. Whereas the structural diversity of DV1Rs progressively increases during gut development up to birth, DV2Rs have limited structural diversity throughout ontogeny. Together, our findings provide evidence for the ability of different fetal tissues to support the development of γδ T cells.