The mammalian middle ear represents one of the most fundamental morphological features that define this class of vertebrates. Its skeletal pattern differs conspicuously from those of other amniotes and has attracted the attention of comparative zoologists for about 200 years. To reconcile this morphological inconsistency, early comparative morphologists suggested that the mammalian middle ear was derived from elements of the jaw joint of nonmammalian amniotes. Fossils of mammalian ancestors also implied a transition in skeletal morphology that resulted in the mammalian state. During the latter half of the 20th century, developmental mechanisms controlling the formation of the jaw skeleton became the subject of studies in developmental biology and molecular genetics. Mammalian middle ear evolution can now be interpreted as a series of changes in the developmental program of the pharyngeal arches. In this review, we summarize the history of middle ear research, highlight some of the remaining problems, and suggest possible future directions. We propose that to understand mammalian middle ear evolution, it is essential to identify the critical developmental events underlying the particular mammalian anatomy and to describe the evolutionary sequence of changes in developmental and molecular terms. We also discuss the degree of consistency between the developmental explanation of the mammalian middle ear based on molecular biology and morphological changes in the fossil record. J. Exp. Zool. (Mol. Dev. Evol.) 314B:417–433, 2010. © 2010 Wiley-Liss, Inc.