Gerbillinae is the second largest subfamily (after Murinae) in the Muridae family comprising ~16 genera and more than a hundred species. Phylogenetic relationships among the main taxa of gerbils are still not fully understood. In particular, a major issue is the phylogenetic position of the monotypic genus Ammodillus (endemic to the Horn of Africa), which according to morphological data may be one of the earliest offshoots from the gerbilline stem; however, its precise affiliations have been unknown due to the lack of genetic data. Here, by means of a multilocus dataset including one mitochondrial and five nuclear markers, we for the first time elucidated phylogenetic placement of Ammodillus and provided the most complete tribal phylogeny of Gerbillinae to date. Phylogenetic reconstructions robustly supported Ammodillus as a sister taxon to all other living gerbillines and suggested that the separation of the ammodile lineage dates back to the Middle/Late Miocene boundary (~11.7 Mya). The results are consistent with subdivision of the subfamily into four tribes: monotypic Ammodillini, Gerbillini (including Taterillus), Desmodilliscini (including Pachyuromys) and Gerbillurini. In the light of the new data, we discuss possible scenarios of Gerbillinae origin, highlight Ammodillus as a relatively ancient—albeit morphologically advanced—lineage that has never gained diversity and propose the term ‘ancient singleton’ for a taxon with a persistently low diversification rate.