OBJECTIVE The objective of this study was to explore the localization of the pylorus, its macroscopic and microscopic development and relationship with neighboring structures. METHODS The study is carried out on 160 human fetuses aged between 9 and 40 weeks of gestation. Abdomen was divided into four quadrants by horizontal and vertical planes passing through the umbilicus. Topographical localization of the pylorus in reference to these quadrants and its distance were determined. Pylorus was divided into pre-pyloric, pyloric, and post-pyloric regions. Starting from the pre-pyloric end, serial sections spanning whole pyloric part were obtained. Wall thickness, the thickness of the muscular coat were measured under light microscope using sections stained with hematoxylin eosin. Sections with the thickest muscular coat were considered as the region where pyloric sphincter was. RESULTS Pylorus was located in the right upper quadrant, on the median plane and in the left upper quadrant. There was a significant relation between the thickness of the muscular coat in the stomach, duodenum and the pyloric region and gestational age. In the region of the pyloric sphincter, the rate of increase in the thickness of the muscular coat was higher in the first and the first half of the second trimesters than term fetuses. CONCLUSIONS We believe that data obtained in the present study will contribute to the assessment of development of the pyloric region in intra-uterine cases.