The X chromosome is known to exert a disproportionately large effect on characters related to post-zygotic reproductive isolation. There is also growing evidence about the important role of the chromosomal regions with reduced recombination (such as inversions) in maintaining the identity of closely related species. Using molecular markers, we examine the effect of different regions of the X chromosome on determination of hybrid traits (viability, testes size, sperm motility and morphological anomalies) in hybrid males between Drosophila madeirensis and Drosophila subobscura. The preponderant effect of a region localized inside the A2 inversion in the X chromosome in all hybrid traits is identified. Other marked regions exert a weaker influence or only influence some of the hybrid trait. Our results confirm the crucial role of sex-linked chromosomal inversion in preserving the identity of species with incomplete reproductive isolation. The specific genomic make-up of parental lines used to perform crosses has a great effect on hybrid fitness.