Abstract – The ashy darter, Etheostoma cinereum, is an imperiled fish within the Cumberland and Tennessee drainages of the southeast United States. An understanding of habitat associations and the relationship of habitat use across multiple spatial scales are critical elements in its conservation. Our objectives were to quantify habitat associations at the stream reach and microhabitat scales for adult and juvenile darters, and to understand the linkage between the two scales based on gradients of habitat use. We focused our efforts within the Rockcastle River, Kentucky (Cumberland River drainage), because the watershed was known to contain a relatively large ashy darter population. Three hundred twenty-two individuals were collected from 21 reaches. The species was restricted to the mainstem of the river and the lower reaches of the larger tributaries. The distribution and abundance of adult and juvenile darters differed significantly at each spatial scale, and both groups demonstrated non-random use of the available habitat. Gradients of stream size and substrate size were identified as important factors. A threshold of environmental quality was determined based on the habitat use patterns among the two scales. Habitat use between the two scales was independent within the threshold, indicating that the specific quality of the microhabitats did not necessarily matter within a stream reach. However, beyond the threshold, a decrease of at least 48% in adult and juvenile darter abundance was seen, indicating that a sufficient network of suitable microhabitats is needed to support a good population of darters within a stream reach.