In eukaryotic cells the genome is highly spatially organized. Functional relevance of higher order genome organization is implied by the fact that specific genes, and even whole chromosomes, alter spatial position in concert with functional changes within the nucleus, for example with modifications to chromatin or transcription. The exact molecular pathways that regulate spatial genome organization and the full implication to the cell of such an organization remain to be determined. However, there is a growing realization that the spatial organization of the genome can be used as a marker of disease. While global genome organization patterns remain largely conserved in disease, some genes and chromosomes occupy distinct nuclear positions in diseased cells compared to their normal counterparts, with the patterns of reorganization differing between diseases. Importantly, mapping the spatial positioning patterns of specific genomic loci can distinguish cancerous tissue from benign with high accuracy. Genome positioning is an attractive novel biomarker since additional quantitative biomarkers are urgently required in many cancer types. Current diagnostic techniques are often subjective and generally lack the ability to identify aggressive cancer from indolent, which can lead to over- or under-treatment of patients. Proof-of-principle for the use of genome positioning as a diagnostic tool has been provided based on small scale retrospective studies. Future large-scale studies are required to assess the feasibility of bringing spatial genome organization-based diagnostics to the clinical setting and to determine if the positioning patterns of specific loci can be useful biomarkers for cancer prognosis. Since spatial reorganization of the genome has been identified in multiple human diseases, it is likely that spatial genome positioning patterns as a diagnostic biomarker may be applied to many diseases.