In 2013, the 29th Chinese National Antarctic Research Expedition (CHINARE) prospected the Prydz Bay on theAntarctic continental shelf, and the Chinese R/V Xuelong icebreaker sampled all of the examined locations. Thenature of Antarctic fish diversity in the high-latitude Prydz Bay is virtually unknown, and the accuracy of relevantestimates has not been established. Thus, it is necessary to evaluate this diversity and propose protectivemeasures. In total, ninety-nine specimens were collected from various locations. To overcome uncertaintiesassociated with identifying species based on morphology, DNA barcoding (COI gene) was employed toreconstruct phylogenetic relationships with delimited references from NCBI. Twenty-two species representing sixfamilies were unambiguously identified from a neighbor-joining (NJ) tree and barcoding gaps. With themorphological identification, thirteen species were identified correctly, five species were identified correctly at thegenus level, and four species were identified at the close sister species level. Notothenioid dominance was notevident in the Prydz Bay, in contrast to other published studies. The low species diversity and catch biomassduring this CHINARE were severely constrained by limited fishing methods and localized sites, which led tobiased underestimation. Our analyses indicate that DNA barcoding is an effective tool for the identification of fishspecies in the Prydz Bay. The identification and distribution of Antarctic fish should be an integral component ofunderstanding Antarctic fish biodiversity and biogeography, and large-scale studies are necessary for the furthertaxonomic identification of Antarctic fish.