The ghost bat, Macroderma gigas, has undergone a major range contraction and is currently restricted around a few, highly disjunct maternity sites. The amount and distribution of mitochondrial DNA (mtDNA) variation within extant populations has been used to assess levels of current and historical maternal gene flow between these populations. An approximately 330 base pair fragment spanning a hypervariable area of the mtDNA control region was amplified and sequenced by using 22 individuals from four current ghost bat populations. The mean sequence diversity of 4.5% between populations was six times higher than that within populations (0.68%), and alleles within populations were monophyletic. Restriction enzyme analysis of amplified products from an additional 100 individuals revealed fixed allelic differences in the distribution of control region genotypes between the four populations. It is suggested that this extreme genetic subdivision is a consequence of long-term female philopatry. For the purposes of management each population should be treated as an independent entity. The depth of the genetic structuring suggests that the isolation of extant populations preceded the historical range contraction.