For any given gene, the identification of exon boundaries and the corresponding flanking intron sequences is the essential prerequisite for asking interesting questions related to its structural organization, spanning from the study of its evolution to the molecular mechanisms underlying possible alternative splicing. In addition, this type of knowledge allows us to carry out mutation screenings for whole exons when the gene in question is known or suspected to be responsible for a human disease phenotype. Several different strategies already used to establish the genomic organization of genes are presented in detail, along with appropriate examples, and they are compared to each other with respect to their requirements, efficiency, and applicability. Particular emphasis is given to the detailed description of the strategy used to study the intron-exon junctions and to establish the intron flanking sequences of the RET proto-oncogene, whose mutations cause Hirschsprung disease (or congenital megacolon) as well as different types of thyroid cancer.