2,7-Diiodo-3,6-dibromofluorene and 2,7-dichloro-3,6-dibromofluorene have been successfully synthesized. The two key intermediates enable us to implement a regioselective Sonogashira reaction followed by intramolecular thiolate/acetylene cyclization, forming two regiospecific pentacyclic dithieno[2,3-b:7,6-b′]fluorene (2,7-DTF) and dithieno[3,2-b:6,7-b′] fluorene (3,6-DTF) isomeric molecules, respectively. By using a similar strategy, selenophene-based diselenopheno[2,3-b:7,6-b′]fluorene (2,7-DSF) as well as diselenopheno[3,2-b:6,7-b′]fluorene (3,6-DSF) were also prepared. The isomeric and sulfur/selenium effects determine the optical, electrochemical, and orbital properties. X-ray crystallography revealed that 2,7-DTF and 3,6-DTF molecules assemble into supramolecular helical structures.