Understanding how chromosomal rearrangements (CRs) interact with epigenetic changes to drive speciation is a fundamental question in evolutionary biology. CRs are key contributors to chromosome evolution and can play a pivotal role in reproductive isolation. The Drosophila nasuta-albomicans species complex presents an ideal model to explore this, as D. albomicans possesses neo-sex chromosomes formed by Robertsonian fusion of chr3L, chr3R, and a sex chromosome, unlike its sister species D. nasuta. In this study, we investigate CRs influence on accessible chromatin (AC) and its relationship with genetic differentiation. We used ATAC-seq to decipher ACs of testis in two hybrids of D. albomicans and D. nasuta, and genome-wide fixation index (FST) scans of D. albomicans and D. nasuta to identify regions of genetic differentiation. Our analyses revealed that chromosome 4 (Muller F) harbors the largest number of differentially accessible regions (~97 Kb), which coincide with peaks in FST. Moreover, changes in ACs were associated with differential transcription factor (TF) binding across the genome. These results suggest that CRs can drive epigenomic divergence in hybrids, particularly on Muller F, and chromatin-level changes may play a key role in reproductive isolation. Our study provides an example of how chromosomal and epigenetic architecture interact in the early stages of speciation.