Pre-mRNA secondary structures are hypothesized to regulate RNA processing pathways, but such structures have been difficult to visualize in vivo. Here, we characterize Saccharomyces cerevisiae pre-mRNA structures through transcriptome-wide dimethyl sulfate probing, enriching for low-abundance pre-mRNA through splicing inhibition. We cross-validate structures found from phylogenetic and mutational studies and identify structures within the majority of measured introns (79 of 88). We find widespread formation of 'zipper stems' between the 5' splice site and branch point, 'downstream stems' between the branch point and the 3' splice site, and previously uncharacterized long stems that distinguish pre-mRNA from spliced mRNA. Multi-dimensional chemical mapping reveals intron structures that independently form in vitro without the presence of binding partners, and structure ensemble prediction suggests that such structures appear in introns across the Saccharomyces genus. We further develop a high-throughput functional assay to characterize variants of RNA structure (VARS-seq), applying it to 135 sets of stems across 7 introns, identifying structured elements that alter retained intron levels at a distance from canonical splice sites. This transcriptome-wide inference of intron RNA structures introduces alternative paradigms and model systems for understanding how pre-mRNA folding influences gene expression.