Regional integration, as an essential measurement for solving unbalanced and uncoordinated regional development, plays an important role in achieving regional sustainable development. In this study, we aimed to construct a systematic research framework to facilitate the development of regional integration. Using 31 prefecture-level cities in the urban agglomeration in the Middle Reaches of the Yangtze River (MYR-UA) as case sites, this study applied box plots, kernel density estimation, GIS visualization tools, Markov chains, and geographic detectors to reveal the dynamic spatiotemporal evolution and factors influencing regional integration from 2009 to 2018. The results indicate that the level of regional integration and its subsystem development is suboptimal in MYR-UA; the temporal characteristic shows an upward fluctuating trend, and the spatial distribution shows remarkable spatial correlation and clustering characteristics. Additionally, we found that the level of regional integration development in MYR-UA has both “path dependence” and “self-locking” effects, and the spatial lag type has a crucial impact on the degree of regional transfer stability. The dominant factors affecting regional integration development include the GDP per capita, economic openness, industrial structure, proportion of education expenditure in fiscal expenditure, urbanization rate, proportion of environmental investment in fiscal expenditure, population density, capital flow, information flow, and technology flow. Finally, based on the findings of this study, policy recommendations for promoting regional integration are proposed.