To fully determine the phylogenetic relationship and ossicle evolution of holothurians, the ossicles of 13 representative sea cucumber species in the China Sea were observed by scanning electronic microscopy and the mitochondrial 16S rDNA gene from eight specimens were amplified and sequenced. The results demonstrated that ossicles of the 13 sea cucumbers could be classified into seven types, which were termed as table, pseudo-table, button, rod, plate, rosette, and oblate grain. The length of 16S rDNA fragments amplified from eight sea cucumbers was approximately 465 bp. These fragments were enriched in AT bases with an average A+T content of 55.8%, which is in accordance with the characteristics of mitochondrial DNA in invertebrates. 16S rDNA sequences of 11 sea cucumbers obtained from GenBank were combined with sequences from our study to construct a phylogenetic tree. was regarded as a control group. Neighbor-joining (NJ) and minimum evolution (ME) methods provided almost identical results. Genetic relationships between in Holothuriidae were rather close and shapes of their ossicles were also similar, thus they could be divided into the same genus; Caudinidae, Phyllophoridae, Sclerodactylidae, and Cucumariidae were clustered with Stichopodidae, and separated from Holothuriidae. Contrary to traditional taxonomy, our study met the criteria of the newest taxonomy system, wherein Aspidochirotida was removed and Holothuroidea was redivided into seven orders, including Dendrochirotida, Synallactida, Molpadida, Persicalida, Holothuriida, Elasipodida, and Apodida. Based on molecular phylogenetic trees and morphological data of ossicles, we hypothesized that ossicles of sea cucumbers in Holothuriidae underwent slow evolution from a complex shape, such as table and button, to simple patterns, such as rosette and oblate grain. Furthermore, considering that the rod in , we concluded that ossicles in Holothuriidae underwent slow transition rather than direct evolution.