Understanding of how the eukaryotic genome is packaged into chromatin and what the functional consequences of this organization are has begun to emerge recently. The concept of ‘chromatin domains’ — the topologically independent structural unit — is the basis of higher order chromatin organization. The idea that this structural unit may also coincide with the functional unit, offers a useful framework in dissecting the structure-function relationship. Boundaries that define these domains have been identified and several assays have been developed to test themin vivo. We have used genetic means to identify and analyse such boundary elements in the bithorax complex ofDrosophila melanogaster. In this review we discuss chromatin domain boundaries identified in several systems using different means. Although there is no significant sequence conservation among various chromatin domain boundaries, these elements show functional conservation across the species. Finally, we discuss mechanistic aspects of how chromatin domain boundaries may function in organizing and regulating eukaryotic genome.