This project provides the genome assembly of the eastern massasauga rattlesnake (Sistrurus catenatus), rSisCat1. The assembly is generated by the Canadian BioGenome Project (https: //www.earthbiogenome.ca/) and CanSeq150 Project provided by CGEn - Canada's national platform for genome sequencing and analysis, and its partners. The data under this project are made available subject to the Canadian BioGenome Project Open Data Release Policy.