A series of conjugation extended donor-acceptor 1,6- and 1,7-regiomers of perylenediimide were synthesized, separated, and characterized. The photophysical, electrochemical, and thermal properties of these compounds were investigated and compared. The absorption spectra of 1,6-substituted PDI showed blue shift as compared to its 1,7-substituted PDI. At the same time, the emission spectrum showed no significant differences among the regiomers. Both 1,6- and 1,7-regiomers were thermally stable up to 450 °C and showed different melting and crystallization transitions. The electrochemical studies did not show significant differences in oxidation and redox potentials owing to similar HOMO/LUMO gap of 1,6- and 1,7-regiomers, which is also supported by theoretical calculations. Comparison of properties of a series of 1,6- or 1,7-substituted PDIs showed significant differences. Such regiomerically pure compounds can offer certain advantages in applications, which are currently being investigated.