Light- and small-angle neutron scattering as well as cryo-transmission electron microscopy (cryo-TEM) studies were performed to probe the structure of J-aggregates formed by a series of achiral dye molecules of the 5,5＇,6,6＇-tetrachlorobenzimidacarbocyanine chromophore having 1,1＇-dialkyl substituents combined with 3,3＇-bis(4-sulfobutyl)-3,3＇-bis(4-carboxybutyl) or 3,3＇-bis(3-carboxypropyl) substituents. Assemblies that display a dependence on the substituents different complex supramolecular structures of nanometer-to-micrometer size have been directly visualized by cryo-TEM. The superstructures span from monomolecular layers formed by the 1,1＇-diethyl-3,3＇-bis(4-sulfobutyl) derivative and stacks of bilayer ribbons in the case of the 1,1＇-dioctyl-3,3＇-bis(4-carboxybutyl) derivative to twisted ropelike structures for the chiral aggregate of the 1,1＇dioctyl-3,3＇-bis(3-carboxypropyl)-substituted chromophore.