Abstract: Often deep genetic divergence indicates potentially different species. However, exceptions are occasionally found. In this study, samples of Opsariichthys bidens from a tributary of the Yellow River (Yiluo River) were analyzed using the mitochondrial cytochrome b (Cyt b) gene, simple sequence repeats (SSR), and the method of stable isotope was used to explore their species status. Phylogenetic trees were reconstructed with the neighbor joining (NJ) and Bayesian inference (BI) methods based on the Cyt b gene sequences and two well supported clades were recovered. Shared haplotype was not detected between these two clades and the average genetic divergence of 3.1% between them seemed to reach the criterion of DNA barcoding for species identification. Nevertheless, analyses of SSR revealed no significant genetic differentiation between the clades (F_st=0.0012, P=1), with 99.88% of variation occurring among individuals. Besides, isotope composition of δ¹³C and δ¹⁵N demonstrated that the two clades shared a common diet with no nutrient niche separation between them. Analysis of SSR and stable isotopes indicated that the two mitochondrially well-separated clades of O. bidens could not be explained by cryptic species, although their deep genetic distances of the Cyt b gene was derived from their ancestral populations.