RNA interference (RNAi) has become one of the most important research tools in functional genomics analysis ever since the discovery of the phenomenon. The robustness of the method has enabled construction of RNAi libraries in the forms of long double-stranded RNA or short-interfering RNA that can cover the whole or significant parts of the genomes of different organisms. Over the last few years, such libraries have been used in different high-throughput formats to establish functional links between genes and phenotypes. In this review, available RNAi library resources and application of these strategic tools will be discussed.