Experimental approaches to define the relationship between gene expression and nuclear matrix attachment regions (MARs) have given contrasting and method‐specific results. We have developed a next generation sequencing strategy to identify MARs across the human genome (MAR‐Seq). The method is based on crosslinking chromatin to its nuclear matrix attachment sites to minimize changes during biochemical processing. We used this method to compare nuclear matrix organization in MCF‐10A mammary epithelial‐like cells and MDA‐MB‐231 breast cancer cells and evaluated the results in the context of global gene expression (array analysis) and positional enrichment of gene‐regulatory histone modifications (ChIP‐Seq). In the normal‐like cells, nuclear matrix‐attached DNA was enriched in expressed genes, while in the breast cancer cells, it was enriched in non‐expressed genes. In both cell lines, the chromatin modifications that mark transcriptional activation or repression were appropriately associated with gene expression. Using this new MAR‐Seq approach, we provide the first genome‐wide characterization of nuclear matrix attachment in mammalian cells and reveal that the nuclear matrix‐associated genome is highly cell‐context dependent. J. Cell. Physiol. 232: 1295–1305, 2017. © 2016 Wiley Periodicals, Inc. MAR‐Seq is a next‐generation sequencing strategy to identify matrix attachment regions (MARs) that is based on crosslinking chromatin to its nuclear attachment sites. Comparison of MCF‐10A mammary epithelial‐like cells and MDA‐MB‐231 breast cancer cells showed that nuclear matrix‐attached DNA in normal‐like MCF‐10A cells is enriched in expressed genes, while in the breast cancer cells, it is enriched in non‐expressed genes. This new MAR‐Seq approach reveals that the nuclear matrix‐associated genome is highly cell‐context dependent.