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Chemical probing experiments interrogate RNA structure by reacting with unpaired residues whose positions are typically read out by mutational profiling on short-read sequencing machines. Such experiments make rapid and genome-wide measurements of RNA structure, but short sequencing reads can rarely be mapped to specific transcript isoforms. Consequently, RNA structure information is acquired as a population average in common regions between transcripts. Here, we present nanopore dimethyl-sulfate mutational profiling (Nano-DMS-MaP-seq) for obtaining isoform resolved structural information of highly similar RNA molecules. We demonstrate the utility of Nano-DMS-MaP-seq by resolving the complex structural landscape of HIV-1 transcripts in infected cells. We show that unspliced and spliced transcripts have distinct structures in the common 5’UTR explaining the specific packaging of only unspliced RNAs into viral particles. Thus, Nano-DMS-MaP-seq is a straightforward method to resolve biologically important transcript-specific RNA structures that were previously hidden in short read ensemble analyses.