Abstract: Hox genes, as a family of transcription factors, play a central role in the specification of regional identities along the anterior-posterior body axis, skeletal development, and limb morphogenesis in vertebrates. Previous analyses of Hox genes revealed an unexpected diversity in gene number, genomic organization, and expression patterns in different vertebrate groups. However, the Hox genes have not been systematically analyzed in terms of sequence and evolutionary features in turtles. In this study, we performed Hox gene identification, sequence characterization and evolutionary analyses in turtles with the publicly available chromosomal-level genome sequences and RNA-seq data, and found that the Hox gene clusters in turtles are highly conserved. Turtles possess the same Hox inventory (39 genes, lost HoxC3) as birds, crocodiles, and placental mammals. The HoxB9-HoxB13 intergenic region in soft-shell turtles is about 10 kb shorter than that in hard-shell turtles, which resulted from the deletion of the non-repetitive sequences. There are hard-shell/soft-shell turtle specific nucleotide substitutions, insertions, and deletions located in the coding regions of tens of Hox genes. However, these sequence mutations are not in functional domains, nor do they lead to changes in protein spatial structure. The Hox genes related to thoracic skeletal development have been undergone rapid evolution and positive selection in the ancestor of soft-shell turtles. The Hox gene expression is tissue- and stage- specific, and mainly expressed in the apical ectoderm ridge, carapace ridge and gonad during the embryonic stages in turtles. We speculate that the phenotypic differences between soft-shell and hard-shell turtles might be related to non-coding regulatory elements located in the HoxB9-HoxB13 intergenic region, and the HoxB5 and HoxC6, which are positively selected in the ancestors of soft-shell turtles. This study provides a target for the analyses of multi-omics, gene expression and regulatory mechanism of Hox genes across different embryonic stages in turtles, also provides a reference for further clarifying the evolutionary innovation in turtles.