The petrous bone is considered the most efficient source of endogenous DNA across skeletal tissues in ancient DNA (aDNA) research as well as in forensic work. Recently, aDNA in auditory ossicle bones was shown to be comparably well preserved as in the petrous, although no attempt was made to distinguish among the three ossicle bones. In this study, we compare aDNA profiles across matched ossicle- and petrous-derived sequencing libraries prepared from 29 human skeletons from Neolithic Anatolia and Medieval Iberia. We find that the stapes and incus provide higher human endogenous aDNA than the petrous bone, with >2× higher median rates of endogenous aDNA recovery, whereas the malleus performs similarly to the petrous. Human aDNA fragments retrieved from the stapes were 8% longer than those from the petrous, whereas postmortem damage, clonality, and contamination rates were comparable among the studied bone types. These observations are corroborated by data from nonmatched ossicle or petrous libraries from 81 individuals from the same contexts, with the highest endogenous aDNA content observed in the stapes. Despite being the smallest bone in the human skeleton, the stapes, along with the incus, may be among the most optimal aDNA sources yet identified.