The evolution of mammalian auditory ossicles was an evolutionary process that resulted in the formation of the mammalian middle ear, where the three middle ear bones or ossicles, namely the incus, malleus and stapes (a.k.a. "the anvil, hammer, and stirrup"), are a defining characteristic of mammals. The event is well-documented and important academically as a demonstration of transitional forms and exaptation, the re-purposing of existing structures during evolution.

The ossicles evolved from skull bones present in most tetrapods, including amphibians, sauropsids (which include extant reptiles and birds) and early synapsids (which include ancestors of mammals). The reptilian quadrate, articular and columella bones are homologs of the mammalian incus, malleus and stapes, respectively. In reptiles (and early synapsids by association), the eardrum is connected to the inner ear via a single bone, the columella, while the upper and lower jaws contain several bones not found in modern mammals. Over the course of mammalian evolution, one bone from the upper jaw (the quadrate) and one from the lower jaw (the articular) lost their function in the jaw articulation and migrated to form the middle ear. The shortened columella connected to these bones to form a kinematic chain of three ossicles, which serve to amplify air-sourced fine vibrations transmitted from the eardrum and facilitate more acute hearing in terrestrial environments.

Following on the ideas of Étienne Geoffroy Saint-Hilaire (1818), and studies by Johann Friedrich Meckel the Younger (1820), Carl Gustav Carus (1818), Martin Rathke (1825), and Karl Ernst von Baer (1828), the relationship between the reptilian jaw bones and mammalian middle-ear bones was first established on the basis of embryology and comparative anatomy by Karl Bogislaus Reichert in 1837. These ideas were advanced by Ernst Gaupp, and are now known as the Reichert–Gaupp theory. Reichert mainly studied the embryological development of the second pharyngeal arch (as in Reichert's cartilage). Despite publishing decades before the publication of On the Origin of Species in 1859, Reichert theorized that part of the reptilian jaw bones and two of the mammalian ossicles are homologous in some sense.

The discovery of the link in homology between the reptilian jaw joint and mammalian malleus and incus is considered an important milestone in the history of comparative anatomy. Work on extinct theromorphs by Owen (1845), and continued by Seeley, Broom, and Watson, was pivotal in discovering the intermediate steps to this change. The transition between the "reptilian" jaw and the "mammalian" middle ear was not bridged in the fossil record until the 1950s with the elaboration of such fossils as the now-famous Morganucodon.

During embryonic development, the incus and malleus arise from the same first pharyngeal arch as the mandible and maxilla, and are served by mandibular and maxillary division of the trigeminal nerve. Recent genetic studies are able to relate the development of the ossicles from the embryonic arch to hypothesized evolutionary history. Bapx1, also known as Nkx3.2 (a member of the NK2 class of homeobox genes), is implicated in the change from the jaw bones of non-mammals to the ossicles of mammals. Other implicated genes include the Dlx genes, Prx genes, and Wnt genes.

Living mammal species can be identified by the presence in females of mammary glands which produce milk. Other features are required when classifying fossils, since mammary glands and other soft-tissue features are not visible in fossils. Paleontologists therefore use the ossicles as distinguishing bony features shared by all living mammals (including monotremes), but not present in any of the early Triassic therapsids ("mammal-like reptiles").

Early amniotes had a jaw joint composed of the articular (a small bone at the back of the lower jaw) and the quadrate (a small bone at the back of the upper jaw). All non-mammalian amniotes use this system including lizards, crocodilians, dinosaurs (and their descendants the birds) and therapsids; so the only ossicle in their middle ears is the stapes. The mammalian jaw joint is composed of different skull bones, including the dentary (the lower jaw bone which carries the teeth) and the squamosal (another small skull bone). In mammals, the quadrate and articular bones have evolved into the incus and malleus bones in the middle ear.

The mammalian middle ear contains three tiny bones known as the ossicles: malleus, incus, and stapes. The ossicles are a complex system of levers whose functions include: reducing the amplitude of the vibrations; increasing the mechanical force of vibrations; and thus improving the efficient transmission of sound energy from the eardrum to the inner ear structures. The ossicles act as the mechanical analog of an electrical transformer, matching the mechanical impedance of vibrations in air to vibrations in the liquid of the cochlea. The net effect of this impedance matching is to greatly increase the overall sensitivity and upper frequency limits of mammalian hearing, as compared to reptilian hearing. The details of these structures and their effects vary noticeably between different mammal species, even when the species are as closely related as humans and chimpanzees.