For the first time direct observation and analysis of microstructural variations of crystalline domains and grain boundaries at atomic scale in the buried interface of an organic semiconductor thin film of poly(2,6-bis(3-alkylthiophen-2-yl)dithieno[3,2-b;2',3'-d]thiophene) (PBTDT), a new synthesized solution-processed polymer is achieved for demonstrating a different network nanostructure of crystalline nanofibers at the interface from the outside surface of the film observed. It is also discovered that structural variations of crystalline domains and grain boundaries at an atomic scale caused by annealing, which include larger domains with enhanced crystallinity, reduced pi-pi, stacking distance, reduced disorders in the grain boundaries, and small tilt-angle boundaries well explain the significant performance improvement of the PBTDT based organic thin film transistor (OTFT) after anealing. This work provides a highly resolutioned image on the microstructures at an organic semiconducting interface for deep scientific insights of the OTFT performance improvement through microstructure optimization.