Covering: up to February 2019Actinomycetes are Gram positive bacteria of the phylum Actinobacteria. These organisms are one of the most important sources of structurally diverse, clinically used antibiotics and other valuable bioactive products, as well as biotechnologically relevant enzymes. Most strains were discovered by their ability to produce a given molecule and were often poorly characterized, physiologically and genetically. The development of genetic methods for Streptomyces and related filamentous actinomycetes has led to the successful manipulation of antibiotic biosynthesis to attain structural modification of microbial metabolites that would have been inaccessible by chemical means and improved production yields. Moreover, genome mining reveals that actinomycete genomes contain multiple biosynthetic gene clusters (BGCs), however only a few of them are expressed under standard laboratory conditions, leading to the production of the respective compound(s). Thus, to access and activate the so-called "silent" BGCs, to improve their biosynthetic potential and to discover novel natural products methodologies for genetic manipulation are required. Although different methods have been applied for many actinomycete strains, genetic engineering is still remaining very challenging for some "underexplored" and poorly characterized actinomycetes. This review summarizes the strategies developed to overcome the obstacles to genetic manipulation of actinomycetes and allowing thereby rational genetic engineering of this industrially relevant group of microorganisms. At the end of this review we give some tips to researchers with limited or no previous experience in genetic manipulation of actinomycetes. The article covers the most relevant literature published until February 2019.