The roll-to-roll manufacturing system is extensively used for mass producing products made of plastic, paper, and fabric in several traditional industries. When flexible substrates, also known as webs, are heated and transported inside the dryer, an inconsistent temperature distribution occurs on the material in the machine direction (MD) and cross-machine direction (CMD). If rollers are not aligned in parallel on the same plane in the roll-to-roll web handling process, or if roller misalignment exists, strain deviation occurs in the web, resulting in lateral displacement and web wrinkles. Therefore, this study examined a wrinkle, which is a thermal deformation that occurs when an inconsistent web temperature distribution is formed on the material inside a dryer. The changes in the elastic modulus and thermal expansion of the web were also examined. Experiments were conducted using a PET film, and its elastic modulus and thermal expansion were examined. The results showed that the presence of a web wrinkle defect can cause a thickness deviation in the functional layer manufactured on the web. Moreover, an appropriate operating speed should be set to reduce the CMD temperature deviation, thereby reducing instances of wrinkle defects.