Polycomb group (PcG) genes encode evolutionarily conserved transcriptional repressors that are required for the long-term silencing of particular developmental control genes in animals and plants. PcG genes were first identified in Drosophila as regulators that keep HOX genes inactive in cells where these genes must remain silent during development. Here, we report the results of a genetic screen aimed at isolating novel PcG mutants in Drosophila. In an EMS mutagenesis, we isolated 82 mutants that show Polycomb-like phenotypes in clones in the adult epidermis and misexpression of the HOX gene Ubx in clones in the imaginal wing disc. Analysis of these mutants revealed that we isolated multiple new alleles in most of the already- known PcG genes. In addition, we isolated multiple mutant alleles in each of ten different genes that previously had not been known to function in PcG repression. We show that the newly identified PcG gene calypso is required for the long-term repression of multiple HOX genes in embryos and larvae. In addition, our studies reveal that the Kto/Med12 and Skd/Med13 subunits of the Med12.Med13.Cdk8.CycC repressor subcomplex of Mediator are needed for repression of the HOX gene Ubx. The results of the mutant screen reported here suggest that the majority of nonredundant Drosophila genes with strong classic PcG phenotypes have been identified.