Reaction of cellular DNA with environmental and chemotherapeutic agents can give rise to a variety of lesions including interstrand cross-links. Because interstrand cross-links can prevent DNA strand separation and thus DNA transcription and replication, they represent a serious impediment to cell survival. Cells have developed mechanisms to repair interstrand cross-links in their DNA and in the case of tumor cells, this can lead to resistance to chemotherapeutic agents. Efforts to investigate the mechanisms by which interstrand cross-links are repaired have been hampered by the difficulty of preparing sufficient quantities of well characterized substrates for physical and biochemical studies. This review will describe synthetic strategies that have been developed to synthesize short DNA oligonucleotide duplexes that contain interstrand cross-links. These short duplexes can be used to study the effects of the cross-link on DNA structure or they can be ligated with larger DNA molecules to produce substrates for repair studies. This review will focus on examples of cross-linked duplexes that have been designed specifically to further our understanding of interstrand cross-link structure and repair.