The organization of homologous protein sequences into multiple sequence alignments (MSAs) is a cornerstone of modern analysis of proteins. Recent focus on the importance of alternatively-spliced isoforms in disease and cell biology has highlighted the need for MSA software that can appropriately account for isoforms and the exon-length insertions or deletions that isoforms may have relative to each other. We previously developedMirage, a software package for generating MSAs for isoforms spanning multiple species. Here, we presentMirage2, which retains the fundamental algorithms of the originalMirageimplementation while providing substantially improved translated mapping and improving several aspects of usability. We demonstrate thatMirage2is highly effective at mapping proteins to their encoding exons, and that these protein-genome mappings lead to extremely accurate intron-aware alignments. Additionally,Mirage2implements a number of engineering improvements that simplify installation and use.