We analyzed changes in the genetic structure of two ecologically distinct populations of Drosophila subobscura by assessing inversion polymorphism parameters for over years. The frequencies of gene arrangements on all five chromosomes show variability in each population. Several chromosomal arrangements appeared for the first time in both populations. Significant difference in the overall distribution of gene arrangements between first and last year of study (1998/2006) in beach wood population of D. subobscura is found for chromosome A (a sex chromosome) and autosomes J and E, while in oak wood population the overall frequency of gene arrangements differ for autosomes J, E and O. The present significant changes of the particular gene arrangements are specific for each population and result in significant differences in comparisons at the level of individual chromosomes between years of study. Although overall heterozygosity remained unchanged over the years in beech habitat, it is significantly reduced in oak population in the fourth year. The obtained results indicate that population structure may significantly depend on short-term and long-term microclimatic changes in habitats. Based on the results to date, monitoring of microevolutionary changes using D. subobscura and its relatives seems a promising way to study the effects of global changes. .