Rosmarinus tomentosus Huber-Morath & Maire, an endemic species of southern Spain, is critically endangered as a consequence of habitat destruction by anthropogenic activities. Random amplified polymorphic DNA (RAPD) markers were used for initial evaluation of genetic variation in this species; among zones, among populations (within zones and independently of zones), and among individuals (within populations and zones). The eight primers used to study five populations provided 126 reliable bands, of which 109 (86%) were polymorphic. Analyses of the RAPD markers conducted using different approaches (cluster analysis, Shannon's diversity measure and analysis of molecular variance-AMOVA) showed similar results concerning genetic structure of populations of R. tomentosus. The three zones studied, and their corresponding populations, were clearly defined through UPGMA analysis. AMOVA analyses indicated that 18.0% of the total genetic variation was attributable to differences among zones. When total variance was partitioned without considering the zonal distribution of the populations, 34.1% was attributable to differences among populations, and 65.9% to differences among individuals within populations. Similar results were obtained using Shannon's information measure. Significant genetic distances (Ph(ist) values) were obtained between any two populations. This strong genetic differentiation among zones, populations and subpopulations may be explained by geographical distances and limited gene flow. This study will be of considerable help during subsequent formulations of conservation strategies for the endangered species R. tomentosus.