The death of the only known female soft-shelled turtle (Rafetus swinhoei) in 2019 marked the imminent danger of the species’ permanent global extinction. To avoid it and facilitate the protection of R. swinhoei, we sequenced and assembled its chromosome-level genome and characterized its sex determination system. Population history analysis indicates R. swinhoei has the lowest heterozygosity among all turtles examined, and its population size has declined dramatically over the past ten million years. Many genes involved in autophagy and DNA damage response were shown to be under positive selection and fast evolution, suggesting their important contributions to the long lifespan and evolutionary adaptation of R. swinhoei. The loss of several tooth-related genes in turtle genomes explains the genetic basis of their toothless phenotype. These results provide genetic insights into the evolution of R. swinhoei, and will be useful for the potential identification of additional female individuals in the field.