Landscape fragmentation due to urban sprawl and industrialization has caused habitat loss and threatened global biodiversity. However, the magnitude, spatial pattern, and mechanisms of how landscape fragmentation leads to habitat loss remain unclear. The Yellow River Basin (YRB) and the Yangtze River Economic Belt (YZREB) are important ecological security barriers that play a decisive role in landscape connectivity and biodiversity conservation in China. From a comparative perspective, this study analyzes the temporal-spatial changes of land cover in the YRB and the YZREB from 2005 to 2018 and simulates the land use pattern in 2031 using a patch-level simulation (PLUS) model. Then the degree and tendency of landscape fragmentation during 2005–2031 are measured based on the entire region and a 10 km grid, respectively. The habitat quality (HQ) is measured using the Integrated Valuation of Ecosystem Services and Tradeoffs (InVEST) model. The temporal-spatial evolution characteristics of HQ are analyzed across multiple dimensions, including the entire region, urban agglomerations and watersheds (upstream, midstream, and downstream). Finally, spatial econometric models are applied to examine the spatial dependence of HQ and quantify the spatial impact of landscape fragmentation on HQ in the YRB and YZREB based on 10 km grids. Results show that: (1) there would be a significant trend of landscape fragmentation and habitat degradation in both the YRB and YZREB during 2005–2031. (2) HQ in the YRB and YZREB has the spatial distribution characteristics of being low in coastal areas and urban agglomerations but high in inland areas and mountainous forest areas. (3) The spatial distribution and spatial relationship of landscape fragmentation and HQ in YRB and YZREB differ significantly in multi-spatiotemporal dimensions. The findings could provide support for landscape planning and biodiversity conservation strategies in China and other developing countries.