Cytological studies revealed that the number of chromosomes and their organization varies across species. The increasing availability of whole genome sequences of multiple species across specific phylogenies has confirmed and greatly extended these cytological observations. In the Drosophila genus, the ancestral karyotype consists of five rod-like acrocentric chromosomes (Muller elements A to E) and one dot-like chromosome (element F), each exhibiting a generally conserved gene content. Chromosomal fusions and paracentric inversions are thus the major contributors, respectively, to chromosome number variation among species and to gene order variation within chromosomal element. The subobscura cluster of Drosophila consists in three species that retain the genus ancestral karyotype and differ by a reduced number of fixed inversions. Here, we have used cytological information and the D. guanche genome sequence to identify and molecularly characterize the breakpoints of inversions that became fixed since the D. guanche-D. subobscura split. Our results have led us to propose a modified version of the D. guanche cytological map of its X chromosome, and to establish that (i) most inversions became fixed in the D. subobscura lineage and (ii) the order in which the four X chromosome overlapping inversions occurred and became fixed.