In order to provide some reference suggestions for promoting the high-quality development of museums in prefecture-level cities and enhancing the regional cultural competitiveness based on an in-depth understanding of the evolutionary characteristics and the influence mechanism of spatial differentiation during the development of museums, 54 museums in Jining (China) were taken as the research object. The study analysed the spatial distribution characteristics of museums in each county and district of Jining and the main influencing factors were explored. The results showed that: (1) museums in Jining tend to be cohesively distributed, mainly concentrated in the central to the northern part of Jining, followed by the north-western part, with a minor distribution in the eastern and southern parts, and the distribution of museums was uneven within Jining; (2) in terms of spatial distribution, museums in Jining have formed a major aggregation area of Qufu-Yanzhou-Rencheng, as well as a sub-aggregation area, centred on Liangshan, and Weishan, Yutai, and other places in southern Jining, although there were museums distributed, compared to the overall distribution, they were fewer in number, smaller in scope, and less evident in aggregation. (3) In terms of influencing factors, the distribution pattern of museums in Jining was related to resource endowment, highway accessibility, and water distribution, i.e., the distribution of museums was more frequent in areas with more natural and historical resources, a developed highway network, and areas near rivers or lakes. The socio-economic situation, the degree of tourism development, and the degree of population density also affected the layout of museums to a certain extent.