Subdivision-based image registration has previously been applied to co-localize digital information extracted from rigid structures in biological specimens, such as the brain. Here, we describe and demonstrate the creation and application of a two-dimensional subdivision-based atlas representing a dynamic structure: the outflow tract of the developing chicken heart. The atlas is designed to segment three different anatomical layers of the outflow tract, and is demonstrated on the characterization of collagen XIV in both control and induced abnormal flow specimens. Abnormal blood flow in the embryonic developing heart can lead to congenital heart disease. Comparing local cellular and sub-cellular changes that are caused by abnormal flow can assist in understanding the molecular pathways involved in maladaptations of the heart and congenital defects. This study demonstrates the approach and potential for more extensive applications of the subdivision-based atlas for the embryonic chicken heart.