Abstract: Using land use data from 1990 to 2020, this study applied the InVEST habitat quality model, integrated with spatiotemporal cube-based hotspot analysis, to examine the spatiotemporal dynamics and spatial heterogeneity of habitat quality in the Nujiang River Basin from temporal, spatial, and geographic perspectives. Results indicate that grassland and forestland dominate the landscape, accounting for 59.7% and 22.0% of the total area, respectively, with land use transitions characterized by a “five increases, four decreases” pattern. Over the past three decades, overall habitat quality remained relatively high (mean = 0.687), but exhibited a gradual decline of 0.009 since 1997. Habitat quality displayed marked spatial variation, forming zonal, dendritic, and reticulated distribution patterns. Low-quality habitats were mainly concentrated in rocky desertification-prone areas in the peripheral upstream south and north, as well as in urbanized zones along the lower reaches. In contrast, high-quality habitats were distributed primarily in mid- and downstream mountainous areas with minimal anthropogenic disturbance, particularly within nature reserves and wetland protection zones. Hotspot analysis revealed significant spatial clustering, with clear cold and hot spot differentiation, closely aligned with habitat quality gradients. These findings offer critical insights for optimizing landscape ecological patterns and support the planning of ecological barrier zones in the Sichuan–Yunnan section of the Yangtze River Economic Belt.