This study presents a quantitative PCR (qPCR) assay for the detection of the endangered diamond darter Crystallaria cincotta from environmental DNA (eDNA) in water samples. The assay design is based on an alignment of mitochondrial cytochrome b DNA sequences from 58 individuals representing 25 percid species. Leveraging genetic differences, a species-specific qPCR assay was designed, incorporating alocked nucleic acid (LNA)-enriched probe and a secondary blocking probe to enhance specificity. The assay targets a 93-base pair fragment that includes a diagnostic single nucleotide polymorphism in the probe region; combined with multiple primer mismatches, this provides specificity for distinguishing C. cincotta from other sympatric percid species. Specificity was validated by testing genomic DNA from 16 percid species and synthetic templates, confirming no cross-reactivity. Performance metrics, including the standard curve, qPCR efficiency, limit of detection, and limit of quantification, are reported. The qPCR assay exhibited sufficient sensitivity to detect C. cincotta eDNA in environmental water samples collected from occupied riverine habitats. This study illustrates the effectiveness of LNA-enriched and blocking probes in developing species-specific qPCR assays for eDNA applications, demonstrating their utility in accurately distinguishing closely related species within diverse fish communities.