Congenital diaphragmatic hernia (CDH) is a frequently occurring birth defect and a source of potentially fatal neonatal respiratory distress. Recently, through the application of detailed karyotyping methods, several CDH-critical regions within the human genome have been identified. These regions typically contain several genes. In this paper we focused on genes from 15q26, the best characterized CDH critical region, as well as FOG2 and GATA4; genes singled out from CDH-critical regions at 8q22-q23 and 8p23.1, respectively. We tested the hypothesis that these putative CDH-related genes are expressed within the developing diaphragm. Our results show that 15q26 contains a cluster of genes that are expressed in the developing rodent diaphragm, consistent with an association between deletions in this region and CDH. Next, we examined the protein expression pattern of positively identified genes, with two major themes emerging. First, those factors strongly associated with CDH are only expressed in the non-muscular, mesenchymal component of the primordial diaphragm, supporting the hypothesis that CDH has its origins in a mesenchymal defect. Second, these factors are all co-expressed in the same cells. This suggests that cases of CDH with unique genetic etiology may lead to a common defect in these cells, and supports the hypothesis that these factors may be members of a common pathway. This study is the first to provide a detailed examination of how genes associated with CDH are expressed in the developing diaphragm, and provides an important foundation for understanding how the deletion of specific genes may contribute to abnormal diaphragm formation.