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The Asian giant soft-shelled turtle, <i>Pelochelys cantorii</i> (Trionychidae), is one of the largest aquatic turtles in China and was designated as a First-Grade Protected Animal in China in 1989. Previous investigation based on a combination of Illumina short-read, PacBio long-read and Hi-C scaffolding technologies acquired a high-quality chromosome-level genome of <i>Pc. cantorii</i>. In this study, comparative genomic analysis between <i>Pc. cantorii</i> and 16 other vertebrate genomes indicated that turtles separated from the ancestor of archosaurians approximately 256.6 (95% highest posterior density interval, 263.6–251.9) million years ago (Mya) (Upper Permian to Triassic) and that <i>Pc. cantorii</i> separated from the ancestor of <i>Pd. sinensis</i> and <i>R. swinhoei</i> approximately 59.3 (95% highest posterior density interval, 64.3–54.3) Mya. Moreover, several candidate genes, such as <i>VWA5A</i>, <i>ABCG2</i>, <i>A2M</i> and <i>IGSF1,</i> associated with tumor suppression, growth and age were expanded, implicating their potential roles in the exceptional longevity of turtles. This new chromosome-level assembly has important scientific value in the study of conservation of <i>Pc. cantorii</i> and also enriches the evolutionary investigation of turtle species.