Chicks hatch with a sterile gastrointestinal tract and receive the first colonization of intestinal bacteria from the shell and hatching environment. Bacteria may translocate from the shell to remaining yolk and/or the developing embryo pre-hatch due to penetration of the protective bloom on the unwashed shell’s surface. The objective of this study was to determine the presence or absence of bacterial DNA via PCR in the gastrointestinal tract of pre-hatch and hatched chicken embryos. Pre-hatch chicken embryo digestive tracts and yolk sacs sampled in a biosafety cabinet on days 13, 20, and 21 of incubation showed no bacterial presence using sensitive 16s PCR. Further research needs to be done in this area, potentially utilizing sterile sampling and cultures to confirm the apparent sterility of the inner egg environment as well as impact of time post-hatch and diversity of bacterial colonization on future production.