The soil-borne and marine gram-positive Actinomycetes are a particularly rich source of carbohydrate-containing metabolites. With the advent of molecular tools and recombinant methods applicable to Actinomycetes, it has become feasible to investigate the biosynthesis of glycosylated compounds at genetic and biochemical levels, which has finally set the basis for engineering novel natural product derivatives. Glycosyltransferases (GT) are key enzymes for the biosynthesis of many valuable natural products that contain sugar moieties and they are most important for drug engineering. So far, the direct cloning of unknown glycosyltransferase genes by polymerase chain reaction (PCR) has not been described because glycosyltransferases do not share strongly conserved amino acid regions. In this study, we report a method for cloning of novel so far unidentified glycosyltransferase genes from different Actinomycetes strain. This was achieved by designing primers after a strategy named consensus-degenerate hybrid oligonucleotide primer (CODEHOP). Using this approach, 22 novel glycosyltransferase encoding genes putatively involved in the decoration of polyketides were cloned from the genomes of 10 Actinomycetes. In addition, a phylogenetic analysis of glycosyltransferases from Actinomycetes is shown in this paper.