Genome-wide measurements of RNA structure can be obtained using reagents that react with unpaired bases, leading to adducts that can be identified by mutational profiling on next generation sequencing machines. One drawback of these experiments is that short sequencing reads can rarely be mapped to specific transcript isoforms. Consequently, information is acquired as a population average in regions that are shared between transcripts thus blurring the underlying structural landscape. Here, we present nanopore dimethyl-sulfate mutational profiling (Nano-DMS-MaP), a method that provides isoform resolved structural information of highly similar RNA molecules. We demonstrate the value of Nano-DMS-MaP by resolving the complex structural landscape of HIV-1 transcripts in infected cells. We show that unspliced and spliced transcripts have distinct structures at the packaging site within their the common 5UTR, likely explaining why spliced viral RNAs are excluded from viral particles. Thus, Nano-DMS-MaP is a straightforward method to resolve biologically important transcript-specific RNA structures that were previously hidden in short read ensemble analyses.