Actinomycetes are a wide group of Gram positive prokaryotes, the soil being their most characteristic habitat, where they play important ecological functions. Their immense biotechnological potential as producers of bioactive molecules of great commercial and industrial interest is exemplified by most antibiotics for clinical use being derived from this group of bacteria. In this work several methodologies and culture media were tested for the isolation of actinomycetes in soils from three different edafoclimatic areas of Tenerife (Canary Islands, Spain): an arid zone of the southeast coast of the island, a humid area in the laurel forest and the Canary high mountain. The results of this work evidenced that just an air drying of the sample during 7 days (pretreatment) produces high counts of actinomycetes versus non-actinomycetes in the three soils and in any of the culture media studied, making unnecessary post physical and chemical treatments. However, the only method that produced an exclusive isolation of actinomycetes was the use of 0.22-μm filters as a physical barrier, and hence being a tool of unquestionable utility to estimate actinomycetes populations in soil.The analysis by BOX rep-PCR determined that the richness and genetic diversity of the isolates of these localities are very high. The greatest similarities were, with one exception, found between strains from the same locations. Izaña was one that obtained the highest diversity. Regarding the biotecnology potential as antibiotic produces isolates from Güimar soils are the most promising as 7 out of 10 isolates showed some antibiotic activity.