Mitochondrial DNA variability of Drosophila subobscura Collin from Southeastern Serbia was studied with respect to Restriction Site Analysis (RSA) of complete mitochondrial genome and the nucleotide sequence of Cytochrome b (Cyt b) gene. The aim was to shed more light on the evolutionary forces that shape mtDNA variation of this species. Samples were collected from two sites in the foothills of the Balkan Mountains. No genetic differentiation was found between groups and most of the variation was observed within them. Restriction analysis revealed two main haplotypes and several rare ones. The sequencing of Cyt b gene showed larger number of haplotypes, among which, one is being the most common. The majority of singletons differed from the most frequent haplotype by one nucleotide change. Although some of the observed nucleotide differences may affect their host?s fitness, the observed pattern of variation is consistent with the seasonal fluctuations in population size.