Although central to evolution, the causes of hybrid inviability that drive reproductive isolation are poorly understood. Embryonic lethality occurs when eggs of the frog X. tropicalis are fertilized with either X. laevis or X. borealis sperm. We observed that distinct subsets of paternal chromosomes failed to assemble functional centromeres, causing their mis-segregation during embryonic cell divisions. Core centromere DNA sequence analysis revealed little conservation among the three species, indicating that epigenetic mechanisms that normally operate to maintain centromere integrity are disrupted on specific paternal chromosomes in hybrids. In vitro reactions combining X. tropicalis egg extract with either X. laevis or X. borealis sperm chromosomes revealed that paternally matched or over-expressed centromeric histone CENP-A and its chaperone HJURP could rescue centromere assembly on affected chromosomes in interphase nuclei. However, whereas the X. laevis chromosomes maintained centromeric CENP-A in metaphase, X. borealis chromosomes did not, and also displayed ultra-thin regions containing ribosomal DNA. Both centromere assembly and morphology of X. borealis mitotic chromosomes could be rescued by inhibiting RNA Polymerase I or by preventing collapse of stalled DNA replication forks. These results indicate that specific paternal centromeres are inactivated in hybrids due to disruption of associated chromatin regions that interfere with CENP-A incorporation, at least in some cases due to conflicts between replication and transcription machineries. Thus, our findings highlight the dynamic nature of centromere maintenance and its susceptibility to disruption in vertebrate interspecies hybrids. Centromere incompatibilities in inviable Xenopus hybrids are sequence-independent and result from disruption of epigenetic pathways required for centromere maintenance.