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The Tehuacán-Cuicatlán Valley, Mexico, is the semiarid region with the richest biodiversity of North America and was recently recognized as a UNESCO’s World Heritage site. Original agricultural practices remain to this day in agroforestry systems (AFS), which are expressions of high biocultural diversity. However, local people and researchers perceive a progressive decline both in natural ecosystems and AFS. To assess changes in location and extent of agricultural land use, we carried out a visual interpretation of very-high resolution imagery and field work, through which we identified AFS and conventional agricultural systems (CAS) from 1995 to 2003 and 2012. We analyzed five communities, representative of three main ecological and agricultural zones of the region. We assessed agricultural land use changes in relation to conspicuous landscape features (relief, rivers, roads, and human settlements). We found that natural ecosystems cover more than 85% of the territory in each community, and AFS represent 51% of all agricultural land. Establishment and permanence of agricultural lands were strongly influenced by gentle slopes and the existence of roads. Contrary to what we expected, we recorded agricultural areas being abandoned, thus favoring the regeneration of natural ecosystems, as well as a 9% increase of AFS over CAS. Agriculture is concentrated near human settlements. Most of the studied territories are meant to preserve natural ecosystems, and traditional AFS practices are being recovered for biocultural conservation.