Agroforestry plays a pivotal role for Sahelian communities by allowing simultaneous improvement of food security and conservation of natural ecosystems and their biodiversity. However, agroforestry systems (AFSs) are particularly heterogeneous in sub-Saharan Africa due to small to very small fields, a large variety of agricultural practices and a diversity of parkland compositions and configurations. This makes spatial sampling processes very important but problematic in terms of representativeness of the landscape heterogeneity to allow an effective study of Sahelian AFSs. In this paper, we proposed, tested and assessed a methodological approach for landscape sampling, mapping and characterization while considering the different types of spatial heterogeneity in complex landscapes, such as Sahelian AFSs. Several complementary methods were combined on the basis of a priori knowledge of agroforestry landscape functioning using multisource data, remote sensing methods, and statistical and spatial analyses applied to landscape ecology. First, the landscape heterogeneity was stratified and used to design two weighted, stratified sampling plans for field surveys of tree species and land use/land cover types. Then, with multisource satellite images together with collected field data, the agroforestry systems were mapped, with a satisfactory accuracy of 85.12% and a Kappa index of 0.81. Finally, we used landscape metrics and diversity indices derived from AFS mapping and the tree species inventory to analyze the diversity of the studied AFS located in the Senegalese Peanut Basin. The results of the analysis evidenced the compositional, configurational and functional heterogeneity found in the study area. This allowed us to demonstrate the ability of the sampling strategy proposed in this paper to capture the various types of heterogeneity in agricultural landscapes. We also showed by implementing the method that it can be used for (i) tree biodiversity analysis, (ii) mapping and (iii) characterization of a complex AFS in sub-Saharan Africa.