Abstract One elusive aspect of the chromosome architecture is how it constrains the DNA topology. Nucleosomes stabilise negative DNA supercoils by restraining a DNA linking number difference (∆Lk) of about −1.26. However, whether this capacity is uniform across the genome is unknown. Here, we calculate the ∆Lk restrained by over 4000 nucleosomes in yeast cells. To achieve this, we insert each nucleosome in a circular minichromosome and perform Topo-seq, a high-throughput procedure to inspect the topology of circular DNA libraries in one gel electrophoresis. We show that nucleosomes inherently restrain distinct ∆Lk values depending on their genomic origin. Nucleosome DNA topologies differ at gene bodies (∆Lk = −1.29), intergenic regions (∆Lk = −1.23), rDNA genes (∆Lk = −1.24) and telomeric regions (∆Lk = −1.07). Nucleosomes near the transcription start and termination sites also exhibit singular DNA topologies. Our findings demonstrate that nucleosome DNA topology is imprinted by its native chromatin context and persists when the nucleosome is relocated.