Smallholder farming landscapes cover large areas of the biodiversity hotspot of the Greater Mekong Subregion and are undergoing rapid land use change driven by economic development, agricultural intensification, and market integration. Yet, their role in maintaining tree diversity, associated ecosystem services, and local knowledge in the ongoing land use transitions remains poorly understood. We assessed perceived on-farm tree species richness, management practices, and species’ cultural importance in upland mixed farming systems, across a land use gradient from extensively managed farms in northern Laos to those intensively managed in northern Vietnam, based on interviews with 371 farmers across five provinces and using species richness as metric for α-, β-, and γ-diversity. The results revealed remarkably high α-diversity (425 tree species), particularly in Laos (297 species), where it was mainly associated with natural regeneration. Vietnam’s smaller and more intensely managed farms, with mainly planted trees, harbored somewhat lower α-diversity but it was partly compensated by higher β-diversity between farms and landscapes. Tree species’ cultural importance and associated knowledge mirrored these patterns, being broadly distributed across multipurpose species in Laos but concentrated around a small number of income-generating species in Vietnam. The proportion of globally threatened species was overall low, but the presence of some threatened taxa in smallholder farming landscapes highlights their potential conservation value. Our findings underscore the importance of supporting natural regeneration, integrating native species into production systems, and strengthening seed and knowledge systems to sustain multifunctional tree diversity in smallholder agricultural mosaics.