Abstract Energy and mineral development, particularly coalbed natural gas development, is proceeding at a rapid pace in the Powder River Structural Basin (PRB) in northeastern Wyoming. Concerns about the potential effects of development led to formation of an interagency working group of primarily Federal and State agencies to address these issues in the PRB in Wyoming and in Montana where similar types of resources exist but are largely undeveloped. Under the direction of the interagency working group, an ecological assessment of streams in the PRB was initiated to determine the current status (2005-06) and to establish a baseline for future monitoring. The ecological assessment components include assessment of stream habitat and riparian zones as well as assessments of macroinvertebrate, algal, and fish communities. All of the components were sampled at 47 sites in the PRB during 2005. A reduced set of components, consisting primarily of macroinvertebrate and fish community assessments, was sampled in 2006. Related ecological data, such as habitat and fish community data collected from selected sites in 2004, also are included in this report. The stream habitat assessment included measurement of channel features, substrate size and embeddedness, riparian vegetation, and reachwide characteristics. The width-to-depth ratio (bankfull width/bankfull depth) tended to be higher at sites on the main-stem Powder River than at sites on the main-stem Tongue River and at sites on tributary streams. The streambed substrate particle size was largest at sites on the main-stem Tongue River and smallest at sites on small tributary streams such as Squirrel Creek and Otter Creek. Total vegetative cover at the ground level, understory, and canopy layers ranged from less than 40 percent at a few sites to more than 90 percent at many of the sites. A bank-stability index indicated that sites in the Tongue River drainage were less at risk of bank failure than sites on the main-stem Powder River. Macroinvertebrate communities showed similarity at the river-drainage scale. Macroinvertebrate communities at sites with mountainous headwaters and snowmelt-driven hydrology, such as Clear Creek, Crazy Woman Creek, and Goose Creek, showed similarity with communities from the main-stem Tongue River. The data also indicated similarity among sites on the main-stem Powder River and among small tributaries of the Tongue River. Data analyses using macroinvertebrate observed/expected models and multimetric indices developed by the States of Wyoming and Montana indicated a tendency toward declining biological condition in the downstream direction along the Tongue River. Biological condition for the main-stem Powder River generally improved downstream, from below Salt Creek to near the Wyoming/Montana border, followed by a general decline downstream from the border to the confluence with the Yellowstone River. The biological condition generally was not significantly different between 2005 and 2006, although streamflow was less in 2006 because of drought. Algal communities showed similarity at the river-drainage scale with slight differences from the pattern observed in the macroinvertebrate communities. Although the algal communities from Clear Creek and Goose Creek were similar to those from the main-stem Tongue River, as was true of the macroinvertebrate communities, the algal communities from Crazy Woman Creek had more similarity to those of main-stem Powder River sites than to the Tongue River sites, contrary to the macroinvertebrates. Ordination of algal communities, as well as diatom metrics including salinity and dominant taxa, indicated substantial variation at two sites along the main stem of the Powder River. Fish communities of the PRB were most diverse in the Tongue River drainage. In part due to the effects of Tongue River Reservoir, 15 species of fish were found in the Tongue River drainage that were not found in the Cheyenne, Belle Fourche, or Little Powder River drainages. The number of introduced species and relative abundance of introduced species of fish were higher in the Tongue River and other drainages than at sites on the main-stem Powder River. Although non- native species were identified in the Powder River, the native fish community is largely intact. Western silvery minnow and sturgeon chub - species of special concern - were identified only at sites on the main- stem Powder River and were most common in the Montana segment of the main stem. Fish and habitat sampling on the main-stem Powder River indicated affinity of some species for certain habitats such as pools, runs, riffles, backwaters, or shoals.