Histone H3 variants play critical roles in the functional specialization of chromatin by epigenetically marking centromeric chromatin and transcriptionally active or silent genes. Specifically, the cenH3 histone variant acts as the primary epigenetic determinant of the site of kinetochore assembly at centromeres. Although the function of histone variants is well studied in plants, animals, and fungi, there is little knowledge of the evolutionary conservation of histone variants and their function in most protists. We find that Giardia intestinalis--a diplomonad parasite with two equivalent nuclei--has two phylogenetically distinct histone H3 variants with N-terminal extensions and nonconserved promoters. To determine their role in chromatin dynamics, conventional H3 and the two H3 variants were GFP-tagged, and their subcellular location was monitored during interphase and mitosis. We demonstrate that one cenH3-like variant has a conserved function in epigenetically marking centromeres. The other H3 variant (H3B) has a punctate distribution on chromosomes, but does not colocalize with active transcriptional regions as indicated by H3K4 methylation. We suggest that H3B could instead mark noncentromeric heterochromatin. Giardia is a member of the Diplomonads and represents an ancient divergence from metazoans and fungi. We confirm the ancient role of histone H3 variants in modulating chromatin architecture, and suggest that monocentric chromosomes represent an ancestral chromosome morphology.