Outer membrane β-barrels (OMBBs) are toroidal arrays of antiparallel β-strands that span the outer membrane of Gram-negative bacteria and eukaryotic organelles. Although homologous, most families of bacterial OMBBs evolved through the independent amplification of an ancestral ββ-hairpin. In mitochondria, one family (SAM50) has a clear bacterial ancestry; the origin of the other family, consisting of 19-stranded OMBBs found only in mitochondria (MOMBBs), is substantially unclear. In a large-scale comparison of mitochondrial and bacterial OMBBs, we find evidence that the common ancestor of all MOMBBs emerged by the amplification of a double ββ-hairpin of bacterial origin, probably at the time of the Last Eukaryotic Common Ancestor. Thus, MOMBBs are indeed descended from bacterial OMBBs, but their fold formed independently in the proto-mitochondria, possibly in response to the need for a general-purpose polypeptide importer. This occurred by a process of amplification, despite the final fold having a prime number of strands.