Background <p>This study investigates the hypothesis that chronic insomnia disorder (CID) is characterized by sex-specific changes in resting-state functional connectivity (rsFC), with certain molecular mechanisms potentially influencing CID's pathophysiology by altering rsFC in relevant networks.</p> Methods <p>Utilizing a resting-state functional magnetic resonance imaging (fMRI) dataset of 395 participants, including 199 CID patients and 196 healthy controls, we examined sex-specific rsFC effects, particularly in the default mode network (DMN) and five insomnia-genetically vulnerable regions of interest (ROIs). By integrating gene expression data from the Allen Human Brain Atlas, we identified genes linked to these sex-specific rsFC alterations and conducted enrichment analysis to uncover underlying molecular mechanisms. Additionally, we simulated the impact of sex differences in rsFC with different sex compositions in our dataset and employed machine learning classifiers to distinguish CID from healthy controls based on sex-specific rsFC data.</p> Results <p>We identified both shared and sex-specific rsFC changes in the DMN and the five genetically vulnerable ROIs, with gene expression variations associated with these sex-specific connectivity differences. Enrichment analysis highlighted genes involved in synaptic signaling, ion channels, and immune function as potential contributors to CID pathophysiology through their influence on connectivity. Furthermore, our findings demonstrate that different sex compositions significantly affect study outcomes and higher diagnostic performance in sex-specific rsFC data than combined sex.</p> Conclusions <p>This study uncovered both shared and sex-specific connectivity alterations in CID, providing molecular insights into its pathophysiology and suggesting considering sex differences in future fMRI-based diagnostic and treatment strategies.</p>