Heterochromatin is integral to cell identity maintenance by impeding the activation of genes for alternate cell fates. Heterochromatic regions are associated with histone 3 lysine 9 trimethylation (H3K9me3) or H3K27me3, but these modifications are also found in euchromatic regions that permit transcription. We discovered that resistance to sonication is a reliable indicator of the heterochromatin state, and we developed a biophysical method (gradient-seq) to discriminate subtypes of H3K9me3 and H3K27me3 domains in sonication-resistant heterochromatin (srHC) versus euchromatin. These classifications are more accurate than the histone marks alone in predicting transcriptional silence and resistance of alternate fate genes to activation during direct cell conversion. Our proteomics of H3K9me3-marked srHC and functional screens revealed diverse proteins, including RBMX and RBMXL1, that impede gene induction during cellular reprogramming. Isolation of srHC with gradient-seq provides a genome-wide map of chromatin structure, elucidating subtypes of repressed domains that are uniquely predictive of diverse other chromatin properties. Display omitted •A physical method allows genomic mapping of heterochromatin that resists sonication•Heterochromatic but not euchromatic H3K9me3 domains impede direct reprogramming•Proteomics reveals 172 proteins enriched in H3K9me3-marked heterochromatin•RBMX and RBMXL1 maintain heterochromatin and resistance to reprogramming Becker et al. present a biophysical method to isolate and map heterochromatin. Genomic regions marked by repressive histone modifications are found in both heterochromatin and euchromatin, with only the former impeding direct cell fate conversion. Proteomics of purified heterochromatin reveals diverse proteins that suppress activation of genes for alternate fates.