The yolk sac and small intestine are 2 important organs responsible for the digestion and absorption of nutrients in chickens during the embryonic and posthatch periods, respectively. The peptide transporter PepT1 is expressed in both the yolk sac and small intestine and plays an important role in the transport of amino acids as short peptides. The objective of this study was to profile the spatial transcriptional patterns of PepT1 mRNA in the yolk sac and small intestine from embryonic and posthatch broilers. The distribution of PepT1 mRNA was investigated by in situ hybridization at embryonic (e) d 11, 13, 15, 17, 19 and day of hatch (doh) in the yolk sac and at e19, doh, and d 1, d 4, and d 7 posthatch in the small intestine. PepT1 mRNA was expressed in the endodermal cells of the yolk sac. PepT1 mRNA was barely detectable at e11, increased from e11 to e13, e15, and e17, and then gradually decreased from e19 to doh. In the small intestine, there was a rapid increase in expression of PepT1 mRNA in the enterocytes from e19 to doh, with expression relatively constant from d 1 to d 7. In addition, there was a differential increase in the heights of the villi in different parts of the small intestine from d 1 to 7, which may partially explain the temporal increase in PepT1 mRNA detected by qPCR. The villi in the duodenum showed the earliest increase in villus height and ultimately resulted in the highest villi at d 7. These results demonstrate that there are temporal changes in PepT1 mRNA expression in the yolk sac and the small intestine, which correspond with their expected role in nutrient uptake during the embryonic and posthatch periods.