To examine the level of genetic differentiation in the sequences of the mitochondrial D-loop gene of Rhinolophus ferrumequinum, and to evaluate the current taxonomic status of this species, 50 tissue samples of greater horseshoe bats were collected in 2011–2015 from 21 different localities in northwest, northeast, west, central, and south regions of Iran. Twenty-two published D-loop sequences from Europe (Switzerland, United Kingdom, Bulgaria, and Tunisia), and Anatolia (south, west, and east Turkey) were downloaded from GenBank. Molecular genetic analyses revealed remarkable variation among populations of R. ferrumequinum. Two major clades with strong support were identified within the greater horseshoe bat. One of these clades consists of individuals of R. ferrumequinum from Iran and eastern Turkey, and is further subdivided into two subclades. A second clade includes samples from western Turkey and Europe. The two subclades from Iran and Turkey and the second clade from western Turkey and Europe represent three diagnosable categories, which most probably warrant three subspecies for the species. Thus, based on genetic differences, it is clear that two subspecific populations are found in Iran: R. f. irani (southern Iran) and R. f. proximus (northern Iran).