The DNA in eukaryotic chromatin is packed by histones into arrays of repeating units called nucleosomes. Each nucleosome contains a nucleosome core, where the DNA is wrapped around a histone octamer, and a stretch of relatively unconstrained DNA called the linker DNA. Since nucleosome cores occlude the DNA from many DNA-binding factors, their positions provide important clues for understanding chromatin packing and gene regulation. Here we review the recent advances in the genome-wide mapping of nucleosome positions, the molecular and structural determinants of nucleosome positioning, and the importance of nucleosome positioning in chromatin higher order folding and transcriptional regulation.