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Analysis of differential alternative splicing from RNA-seq data is complicated by the fact that many RNA-seq reads map to multiple transcripts, and that annotated transcripts from a given gene are often a small subset of many possible complete transcripts for that gene. Here we describe Yanagi, a tool which segments a transcriptome into disjoint regions to create a segments library from a complete transcriptome annotation that preserves all of its consecutive regions of a given length L while distinguishing annotated alternative splicing events in the transcriptome. In this paper, we formalize this concept of transcriptome segmentation and propose an efficient algorithm for generating segment libraries based on a length parameter dependent on specific RNA-Seq library construction. The resulting segment sequences can be used with pseudo-alignment tools to quantify expression at the segment level. We characterize the segment libraries for the reference transcriptomes of Drosophila melanogaster and Homo sapiens. Finally, we demonstrate the utility of quantification using a segment library based on an analysis of differential exon skipping in Drosophila melanogaster and Homo sapiens. The notion of transcript segmentation as introduced here and implemented in Yanagi will open the door for the application of lightweight, ultra-fast pseudo-alignment algorithms in a wide variety of analyses of transcription variation.