This study investigates the population genetic structure of Miniopterus pallidus using the D-loop region of mitochondrial DNA (331 bp) from 13 localities in Iran. We found a high number of haplotypes (N = 43) and high nucleotide diversity (π = 0.02). The average K2P genetic distance was high within (2.59%) and between (2.54%) the Iranian populations of M. pallidus and analysis of molecular variance revealed that most genetic variability was related to differences within populations. The positive and significant association between pairwise genetic and environmental distances (r = 0.443, p = .01) after accounting for the effect of geographical distances suggests that substantial influence of the climatic factors among different sites have shaped the genetic variation in this species. Neutrality tests showed historical demographic events when applying Fu’s Fs, but Tajima’s statistic was nonsignificant, indicating a deviation from the theoretical model of expansion. The hypothesis of sudden-expansion was not rejected by analyses of mismatch distribution as the Raggedness and SSD were insignificant (p(r)= .39 and p(SSD) = .38). Similarly, BSP results approximately revealed a major historical expansion around 81,000 years ago and a recent population decline around 13,000 years ago. Phylogenetic trees illustrated that all Iranian M. pallidus populations, as well as those from eastern Turkey and Azerbaijan, form a monophyletic clade, whereas the samples from western Turkey, Georgia and Greece belong to M. schreibersii clade. The haplotype network was consistent with these findings for M. pallidus populations in Iran and showed a high level of reticulation.