The Niangua darter, Etheostoma nianguae, is a threatened stream fish endemic to the Osage River basin in south-central Missouri. We studied the darter's distributional patterns at three spatial scales (stream, reach, and microhabitat) to assist ongoing conservation efforts. Darter presence-absence as a function of one or more habitat variables was modeled with logistic regression at each scale. The most important predictors of presence-absence were stream link magnitude, mean confluence difference (CD), reach length or gradient, streambed elevation, bank erosion index, water depth, and substrate mean particle size. At the stream scale, darters were present in larger streams (e.g., fifth order) with small CDs. A relatively small CD, for example, described the convergence of two streams of roughly equal size in the drainage basin, whereas a relatively large CD indicated a substantial difference at a confluence. We propose that the CD metric represents a large-scale zoogeographic barrier to E. nianguae, excluding this species from any tributary stream that flows into a receiving stream that is three or more stream orders larger than the tributary. Within one occupied stream, the Little Niangua River, darters were found disproportionately in reaches (1) located in the mid- to lower sections of the stream (elevations 230–250 m above sea level), (2) with riffles spaced 40–80 m apart or with gradients of 2–4 m × km⁻¹, and (3) with relatively uneroded banks. Within occupied reaches, they were commonly located in microhabitats 20–40 cm deep with substrate particles averaging 30–50 mm in diameter. Multivariate model precision ranged from 29–57% within single scales. The models can be used to guide conservation and recovery efforts by ranking sites in the Osage basin based on their relative suitability for E. nianguae.