Agricultural cultural heritage is crucial in advancing comprehensive rural revitalization. The Yangtze River Basin is rich in biodiversity and abundant in fishery cultural resources. The cultural resources not only reflect the ecological wisdom of harmonious coexistence and the human–land relationship between humans and nature but also provide critical cultural support for rural revitalization and watershed sustainable development. This study investigates the spatial distribution, influencing factors, and historical evolution of fishery cultural resources in the Yangtze River Basin. The highest proportions of significant resources are found in fishery engineering and landscapes, culinary flavors, fishery customs, dances, fishery gear, and poetry. By analyzing 14 categories of fishery cultural resources and 157 national and provincial intangible cultural heritage items, this study reveals a significant geographical clustering of these resources in the mid-lower reaches, particularly around the middle reaches of the Yangtze River and Poyang Lake Basin, Wuhan and Dongting Lake, and the lower reaches of the Taihu Lake Basin. This study underscores the role of natural geographical conditions, aquatic biodiversity, socio-economic factors, and historical–cultural backgrounds in the distribution and evolution of fishery cultural resources, with these factors interacting dynamically. By advocating for a comprehensive approach to coordinating fishery culture preservation with rural revitalization, this study outlines a multidimensional strategy for the preservation and sustainable development of fishery cultural resources.