Heterochromatin protein 1 (HP1) is a well-characterized heterochromatin component conserved from fission yeast to humans. We identified three HP1-like genes (hcpA, hcpB, and hcpC) in the Dictyostelium discoideum genome. Two of these (hcpA and hcpB) are expressed, and the proteins colocalized as green fluorescent protein (GFP) fusion proteins in one major cluster at the nuclear periphery that was also characterized by histone H3 lysine 9 dimethylation, a histone modification so far not described for Dictyostelium. The data strongly suggest that this cluster represents the centromeres. Both single-knockout strains displayed only subtle phenotypes, suggesting that both isoforms have largely overlapping functions. In contrast, disruption of both isoforms appeared to be lethal. Furthermore, overexpression of a C-terminally truncated form of HcpA resulted in phenotypically distinct growth defects that were characterized by a strong decrease in cell viability. Although genetic evidence implies functional redundancy, overexpression of GFP-HcpA, but not GFP-HcpB, caused growth defects that were accompanied by an increase in the frequency of atypic anaphase bridges. Our data indicate that Dictyostelium discoideum cells are sensitive to changes in HcpA and HcpB protein levels and that the two isoforms display different in vivo and in vitro affinities for each other. Since the RNA interference (RNAi) machinery is frequently involved in chromatin remodeling, we analyzed if knockouts of RNAi components influenced the localization of H3K9 dimethylation and HP1 isoforms in Dictyostelium. Interestingly, heterochromatin organization appeared to be independent of functional RNAi.