Organic thin-film semiconductor devices have important research and application value in the fields of microelectronics and optoelectronics. The film quality is one of the key factors affecting the device performance such as the uniformity of film spatial distribution. This parameter was studied by analyzing the reflectance difference maps of pentacene thin film grown on an anisotropic substrate which were measured by reflectance difference microscopy. The results demonstrate the capability of reflectance difference microscopy to study the processing of thin film growth.