Dictyostelid species classification has traditionally relied on morphology, a time-intensive method requiring expert knowledge. This study evaluated the potential and limitations of using the 18S rDNA sequence for species-level classification. 18S rDNA sequences of 16 samples from the Dicty stock center, including 14 samples found in Thailand, were analyzed. Signature sequence analyses confirmed genus-level identification with high accuracy. These sequences were analyzed alongside 309 database entries retrieved from the GenBank database. The analyses confirmed genus-level identification accuracy but highlighted challenges in distinguishing species due to overlapping intraspecific and interspecific variations, negative barcoding gaps, and incorrectly grouped samples to putative taxa by species delimitation analyses. Species delimitation methods, including maximum likelihood (ML) phylogenetic analysis, achieved limited success, with ML showing the highest accuracy but not exceeding 50%. However, species with high barcoding gaps, such as Raperostelium and Rostrostelium, demonstrated potential for accurate classification. These findings support using 18S rDNA for genus-level identification and suggest its possible application for certain species. Expanded sampling is needed to improve species-level classification and to identify more robust DNA markers for dictyostelid diversity studies.