Three diacetylene-containing peptide amphiphiles, with exactly the same structure, except for the position of the diacetylene moiety within the hydrophobic tail, were investigated on their assembly behavior, polymerization characteristics, and chromatic properties. After polymerization it was found that the color and, thus, the conjugation length of the polydiacetylenes was dependent both on mobility and on packing of the groups on both sides of the triple bonds. The polydiacetylene with the highest conjugation length and most stable structure was obtained when the diacetylenes were positioned in the middle of the hydrophobic tail. When the diacetylenes were close to the peptide, the mismatch between hydrogen bond and alkyl tail spacing resulted in a decreased conjugation length of the resulting polymer, while when positioned close to the end of the amphiphile the mobility of the terminal alkyl chains hampered the packing of the diacetylenes.