The axolotl (Ambystoma mexicanum) provides critical models for studying regeneration, evolution, and development. However, its large genome (near ten times larger than the human) presents a formidable barrier to genetic analyses. A new assembly encompasses 94% of annotated gene models on chromosomal scaffolds. The assembly’s utility was demonstrated by resolving genome-wide orthologues between the axolotl and other vertebrates, identifying the footprints of historical introgression events that occurred during the development of axolotl genetic stocks, and precisely mapping several phenotypes including a large deletion underlying the cardiac mutant (thus providing a new model of human disease). This chromosome-scale assembly will greatly facilitate studies of the axolotl in biological research, especially regenerative medicine.