The nucleosome is a structural and functional subunit of chromatin, and its positioning and dynamics in the eukaryotic genomes serves as a key platform for gene regulation. Here, we determine the positions of fully wrapped nucleosomes across the yeast genome by chemical mapping through the histone H2A-A122C residue, which cleaves near the DNA entry/exit sites. This approach reveals the most refined sequence-dependent profile of nucleosomes reported to date. Comparison of H2A-A122C with the H3-Q85C and H4-S47C methods clearly shows a sequence preference for the chemical cleavage site, and mapping results suggest that the nucleosome locations are dynamic in vivo. More importantly, we find that the depletion of CC and GG dinucleotides at nucleotide positions -11 to -9 and +9 to +11 bp, respectively, from the nucleosome dyad position (0) is inextricably associated with the enrichment of AA/AT/TA/TT dinucleotides in both yeast and mouse genomes. Introducing consecutive C{middle dot}G base pairs to the corresponding sites in the 601 sequence shifts the nucleosomes to less frequent positions without altering thermal stability, implying a structural constraint imposed by DNA sequence. Thus, CC and GG dinucleotides in the major groove blocks at superhelix locations (SHL) -1.0 and +1.0, respectively, destabilize histone-DNA interactions, serving as intrinsic determinants of nucleosome positioning in eukaryotic genomes.