Freshwater fishes of the superfamily Cobitoidea are a species-rich group that are present in virtually every river in Europe and Asia. Three of the nine recognized families show massive karyotype reconstructions, including several independent cases of polyploidy, indicating cytogenetic evolution correlated with their diversification. We herein present the karyotype of a member of the monogeneric family Ellopostomatidae, a very rarely collected and morphologically highly enigmatic Southeast Asian representative of the Cobitoidea. The analyzed specimen of Ellopostoma mystax from southern Thailand had a diploid chromosome number of 2n = 48, with karyotype composed of three pairs of metacentric (m), 19 pairs of submeta- to subtelocentric (sm, st), and two pairs of acrocentric (a) chromosomes. Six pairs of sm to st chromosomes bore CMA₃-positive sites either in pericentromeric positions or formed CMA₃-positive p arms of the carrier chromosomes. Nucleolar organizing regions (NORs) as visualized by silver (Ag) impregnation were situated on the entire length of the shorter arms of the second largest sm to st pair of chromosomes; these corresponding sites also stained CMA₃-positive. When compared with other families of Cobitoidea, the karyotype of Ellopostoma reveals a rather plesiomorphic composition, suggesting that the strong derivation of Ellopostoma based on morphology only was not accompanied by a cytogenetic derivation.