Background: Chromatin architecture is an essential factor regulating gene transcription in different cell types and developmental phases. However, studies on chromatin architecture in perennial woody plants and on the function of chromatin organization in sex determination have not been reported. Results: Here, we produced a chromosome-scale de novo assembly of the woody plant Jatropha curcas genome with a total length of 379.5 Mb and a scaffold N50 of 30.7 Mb using PacBio long reads combined with genome-wide chromosome conformation capture (Hi-C) technology. Based on this high-quality reference genome, we detected chromatin architecture differences between monoecious and gynoecious inflorescence buds of Jatropha. Differentially expressed genes (DEGs) were significantly enriched in the changed A/B compartments and topologically associated domain (TAD) regions and occurred preferentially in differential contact regions between monoecious and gynoecious inflorescence buds. Twelve DEGs related to flower development or hormone synthesis displayed significantly different genomic interaction patterns in monoecious and gynoecious inflorescence buds. These results demonstrate that chromatin organization participates in the regulation of gene transcription during the process of sex differentiation in Jatropha. Conclusions: We have revealed the features of chromatin architecture in perennial woody plants and investigated the possible function of chromatin organization in Jatropha sex differentiation. These findings will facilitate understanding of the regulatory mechanisms of sex determination in higher plants.