Sea cucumbers are ecologically and economically significant marine organisms, but accurately identifying their species remains a challenge due to morphological similarities and the presence of cryptic species. This issue is particularly pronounced in regions viz. West Nusa Tenggara (NTB) and East Nusa Tenggara (NTT), Indonesia, where high biodiversity and overexploitation pressures necessitate precise species identification for effective management and conservation. This study aimed to evaluate the effectiveness of DNA barcoding and phylogenetic analysis in identifying sea cucumber species in these regions. A total of ten sea cucumber samples were collected from various locations in NTB and NTT and were analyzed using the mitochondrial COI gene as a barcode marker. PCR amplification and sequencing were performed, followed by BLAST analysis and phylogenetic tree construction using the maximum likelihood method. The results indicated that eight samples were successfully identified at the species level, with percent identity ranging from 96.39 to 100%. This confirmed the high accuracy of DNA barcoding in distinguishing sea cucumber species. Notably, Stichopus monotuberculatus was identified with 100% identity, while Holothuria flavomaculata showed slightly lower identity values, suggesting potential intraspecific genetic variation or the presence of cryptic species. Phylogenetic analysis further supported these findings, revealing distinct clades among the identified species and indicating significant genetic divergence within the studied populations. However, limitations such as incomplete reference databases and suboptimal sequence quality hindered the identification of two samples. These findings emphasize the need to expand genetic reference data and improve sequence quality to enhance the reliability of DNA barcoding. Overall, this study demonstrates the utility of molecular approaches in addressing species identification challenges, contributing to the sustainable management and conservation of sea cucumber populations in NTB and NTT.