We　present　a　method　for　multiplexed　in　situ　imaging　of　individual　RNA　splicing　variants.　This　method　enables　quantifying　RNA　splicing　variants　with　single-molecule　resolution,　discriminating　splicing　isoforms　with　single-base　precision　as　well　as　analyzing　the　subcellular　localization　of　transcripts.　With　this　technology,　it　is　possible　to　study　cell　heterogeneity　of　gene　expression,　potentially　helping　to　decipher　rich　diversity　in　posttranscriptional　function　and　assist　clinical　monitoring.　©　2022,　The　Author(s),　under　exclusive　license　to　Springer　Science+Business　Media,　LLC,　part　of　Springer　Nature.