Eukaryotic DNA is wound around histone proteins to form repeating units of nucleosomes that are packaged into higher order structures to form chromosomes. Nucleosomal packaging is dynamically regulated to control the expression of the genome through regulating access of transcription factors to specific genes in response to stimuli. Critical for these processes are modifications to the N-terminal tails of histones. In particular, methylation of specific histone lysine residues has a profound impact on transcription. This “mark” recruits protein complexes that contain methyl-lysine “reader” modules that bind to the mark and facilitate transcriptional silencing or activation of the underlying gene. Mutation and misregulation of proteins that “write,” “read” and “erase” these marks play a foundational role in the development and progression of nearly all cancers. Chemical tools that antagonize these writers, readers and erasers have been developed in recent years. These tools have been valuable in explicating the biology of these proteins, and in some cases, as therapeutic agents. Development of antagonists of methyl-lysine readers has lagged compared to both methyl-lysine writers and erasers. Herein we describe the design, development and characterization of antagonists of the chromodomain reader modules contained in Polycomb Repressive Complex I (PRC1). PRC1 contains one of five chromodomains; CBX2, -4, -6, -7 or -8. PRC1 complexes containing any one of these proteins play critical roles in normal, growth and development. Accordingly, misregulation of PRC1 chromodomains contributes to the initiation and development of numerous cancers. We developed a series of potent, peptidic antagonists of PRC1 chromodomains and an expanded set of chemical tools around one of these molecules, UNC3866, which has the highest affinity for the chromodomains of CBX4 and -7. We demonstrated the ability of UNC3866 to inhibit proliferation in a metastatic prostate cancer cells through the induction of a senescent-like state. Further optimization of UNC3866 led to a second-generation antagonist of PRC1 chromodomains with a dramatically improved residence time for CBX7 and increased cellular potency. This work has produced a valuable tool set for studying CBX biology and laid the ground work for continued efforts to evaluate and improve CBX antagonists as potential therapeutic agents.