Summary Understanding the evolutionary conservation of complex eukaryotic transcriptomes significantly illuminates the physiological relevance of alternative splicing (AS). Examining the evolutionary depth of a given AS event with ordinary homology searches is generally challenging and time‐consuming. Here, we present C atsnap , an algorithmic pipeline for assessing the conservation of putative protein isoforms generated by AS. It employs a machine learning approach following a database search with the provided pair of protein sequences. We used the C atsnap algorithm for analyzing the conservation of emerging experimentally characterized alternative proteins from plants and animals. Indeed, most of them are conserved among other species. C atsnap can detect the conserved functional protein isoforms regardless of the AS type by which they are generated. Notably, we found that while the primary amino acid sequence is maintained, the type of AS determining the inclusion or exclusion of protein regions varies throughout plant phylogenetic lineages in these proteins. We also document that this phenomenon is less seen among animals. In sum, our algorithm highlights the presence of unexpectedly frequent hotspots where protein isoforms recurrently arise to carry physiologically relevant functions. The user web interface is available at https://catsnap.cesnet.cz/ .