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Summary The DNA of eukaryotes is wrapped around histone octamers to form nucleosomes. Although it is well established that the DNA sequence significantly influences nucleosome formation, its precise contribution has remained controversial, partially owing to the lack of quantitative affinity data. Here, we present a method to measure DNA-histone binding free energies at medium throughput and with high sensitivity. Competitive nucleosome formation is achieved through automation, and a modified epifluorescence microscope is used to rapidly and accurately measure the fractions of bound/unbound DNA based on fluorescence anisotropy. The procedure allows us to obtain full titration curves with high reproducibility. We applied this technique to measure the histone-DNA affinities for 47 DNA sequences and analyzed how the affinities correlate with relevant DNA sequence features. We found that the GC content has a significant impact on nucleosome-forming preferences, but 10 bp dinucleotide periodicities and the presence of poly(dA:dT) stretches do not., Graphical Abstract, Highlights • Robotics permits full titration series to measure histone-DNA binding affinities • Fluorescence anisotropy used as a fast, sensitive readout of bound/unbound DNA • Free energies span three orders of magnitude, less for naturally occurring sequences • GC content is a major determinant of measured binding free energies, Molecular Biology; Molecular Interaction; Biological Sciences Research Methodologies