A detailed analysis of the optical and photophysical properties of 2,2‘:5‘:2‘ ‘:5‘ ‘,2‘ ‘‘-quaterthiophene (QT), 3,3‘ ‘‘-dimethoxy-2,2‘:5‘:2‘ ‘:5‘ ‘,2‘ ‘‘-quaterthiophene (DMOQT), 3,3‘ ‘‘-dimethyl-2,2‘:5‘:2‘ ‘:5‘ ‘,2‘ ‘‘-quaterthiophene (DMQT) and 3‘,4‘ ‘-didecyl-2,2‘:5‘:2‘ ‘:5‘ ‘,2‘ ‘‘-quaterthiophene (DDQT) in various environments is reported. In solution at room temperature, the optical properties of the free molecules are obtained and discussed in terms of the effect of the substitution on the conformation adopted by each derivative in the electronic ground and first excited states. In a tetradecane matrix at 77 K, the optical properties are obtained for the isolated molecules in this rigid medium where the oligothiophenes adopt conformations similar to those found in the solid state. The optical properties of the quaterthiophene derivatives in their aggregated forms and in the solid state are also reported and discussed in terms of the substitutional effect on the intermolecular interactions, which affect the spectral and photophysical properties of the isolated molecules. For the first time, a β,β‘-disubstituted oligothiophene (DMQT) showing an excitonic splitting similar to that obtained for QT is reported. All other substituted oligothiophenes presented show a conformational change, following the aggregation process. This difference is explained by more disordered crystalline forms for DMOQT and DDQT. Theoretical calculations using the ZINDO/S semiempirical method are also performed on the crystalline structure of each derivative in an attempt to correlate the optical properties of these molecules in their aggregated forms and in the solid state with the molecular arrangement found in the crystal.