In human cells, Haspin-mediated histone H3 threonine 3 phosphorylation (H3T3ph) promotes centromeric localization of the Chromosome Passenger Complex, thereby ensuring proper kinetochore–microtubule attachment. Haspin also binds to PDS5 cohesin associated factor B (Pds5B), antagonizing the Wings apart-like protein homolog (Wapl) –Pds5B interaction and thus preventing Wapl from releasing centromeric cohesion during mitosis. However, the role of Haspin in plant chromosome segregation is not well understood. Here, we show that in maize (Zea mays) mitotic cells, ZmHaspin localized to the centromere during metaphase and anaphase while localizing to the telomeres during meiosis. These results suggest that ZmHaspin plays different roles during mitosis and meiosis. ZmHaspin knockout led to decreased H3T3ph and H3S10ph and defects in chromosome alignment and segregation in mitosis. These lines of evidence suggest that Haspin regulates chromosome segregation in plants via the mechanism described for humans, namely, H3T3ph. Plant Haspin proteins lack the RTYGA and PxVxL motifs needed to bind Pds5B and HP1 and no obvious cohesion defects were detected in ZmHaspin knockout plants. Taken together, these results highlight the conserved but slightly different roles of Haspin proteins in cell division in plants vs. animals.