Poly(thiophene)s have an inherently large third-order nonlinear optical (TONO) response, but applications are not straightforward due to unoptimized materials. Therefore, several structure-property relationships (molar mass, branching, regioregularity) are investigated to unravel which structural modifications give the highest TONO response. Poly(3-hexylthiophene) with different molar masses, poly[3-(2-ethylhexyl)thiophene] with different molar masses, and random copolymers with different degrees of regioregularity are synthesized and measured by UV-vis spectroscopy and the third harmonic scattering technique. Every structural modification that leads to an increase in π-π interactions in poly(thiophene)s leads to an increase in the TONO response of the material. Therefore, a material with a high molar mass, an unbranched alkyl side chain, and a high regioregularity degree is preferably tested as a promising TONO material.