Numerous long non-coding RNAs (lncRNAs) have emerged as having important and mechanistically diverse roles in gene regulation. However, for most lncRNAs, evolution is rapid and functions remain uncharacterized. This Review describes how comparative genomics and transcriptomics can provide broad insights into the functional importance and evolutionary trajectories of lncRNAs across species, and offers guidance for cross-species studies of lncRNAs.