Abstract: The structures of RNA molecules are often important for their function and regulation, yet there are no experimental techniques for genome-scale measurement of RNA structure. Here we describe a novel strategy termed parallel analysis of RNA structure (PARS), which is based on deep sequencing fragments of RNAs that were treated with structure-specific enzymes, thus providing simultaneous in vitro profiling of the secondary structure of thousands of RNA species at single nucleotide resolution. We apply PARS to profile the secondary structure of the messenger RNAs (mRNAs) of the budding yeast Saccharomyces cerevisiae and obtain structural profiles for over 3,000 distinct transcripts. Analysis of these profiles reveals several RNA structural properties of yeast transcripts, including the existence of more secondary structure over coding regions compared with untranslated regions, a three-nucleotide periodicity of secondary structure across coding regions and an anti-correlation between the efficiency with which an mRNA is translated and the structure over its translation start site. PARS is readily applicable to other organisms and to profiling RNA structure in diverse conditions, thus enabling studies of the dynamics of secondary structure at a genomic scale.