Approaches linking biodiversity assessment with landscape structure are necessary in the framework of sustainable rural development. The present paper describes a methodology to estimate plant diversity involving landscape structure as a proportional weight associated with different plant communities found in the landscape mosaic. The area occupied by a plant community, its patch number or its spatial distribution of patches are variables that could be expressed in gamma plant diversity of a territory. The methodology applies (1) remote sensing information, to identify land cover and land use types; (2) aspect, to discriminate composition of plant communities in each land cover type; (3) multi-scale field techniques, to asses plant diversity; (4) affinity analysis of plant community composition, to validate the stratified random sampling design and (5) the additive model that partitions gamma diversity into its alpha and beta components. The method was applied to three Spanish rural areas and was able to record 150–260 species per ha. Species richness, Shannon information index and Simpson concentration index were used to measure diversity in each area. The estimation using Shannon diversity index and the product of patch number and patch interspersion as weighting of plant community diversity was found to be the most appropriate method of measuring plant diversity at the landscape level.