The Seto Inland Sea is one of the largest semi-enclosed seas of Japan, where inflows of both nutrient-rich freshwater and nutrient-poor open ocean water have a profound influence on oceanographic conditions in the surface layers. To explore the diversity and distribution of unicellular diazotrophic cyanobacteria in this highly complex coastal area, we evaluated the seasonal dynamics of the cyanobacteria in the Seto Inland Sea over a period of 2 years. We obtained sequences belonging to UCYN-A and UCYN-C from the UCYN 16S rRNA gene clone library. The UCYN-C sequences were divided into three major types, referred to as UCYN-C ribotypes 1, 2, and 3; these groups clustered with Cyanothece ATCC51142, Cyanothece SKTU126, and the endosymbiont of Rhopalodia gibba, respectively. Results of qPCR showed that UCYN-A was detected during the early summer season at sampling points with temperatures ranging from 14 to 28 °C and with relatively nitrogen-depleted conditions. The highest abundance of UCYN-C was observed during the winter season, reaching 8.3 × 105 nifH gene copies l−1. Our results revealed a widespread presence of unicellular diazotrophic cyanobacteria in the Seto Inland Sea, suggesting their contribution to the biogeochemical cycle.