We introduce a k-mer-based computational protocol, DE-kupl, for capturing local RNA variation in a set of RNA-seq libraries, independently of a reference genome or transcriptome. DE-kupl extracts all k-mers with differential abundance directly from the raw data files. This enables the retrieval of virtually all variation present in an RNA-seq data set. This variation is subsequently assigned to biological events or entities such as differential long non-coding RNAs, splice and polyadenylation variants, introns, repeats, editing or mutation events, and exogenous RNA. Applying DE-kupl to human RNA-seq data sets identified multiple types of novel events, reproducibly across independent RNA-seq experiments.