Between October 2001 and December 2002 the researchers sampled sites along the headwaters of the Roanoke River in Southwestern Virginia. These upper headwaters provide the drinking-water supply for towns and cities downstream. Sampling sites were included on both the North Fork and the Roanoke Main Stem. Benthic habitat, physical conditions (including watershed geology), macroinvertebrates, and diatoms from the two branches were analyzed and compared. Benthic macroinvertebrates were sorted and identified with analysis of total number of taxa and EPT. The experiment showed significant differences in macroinvertebrates between the two steams, possibly associated with land use. Taxa diversity was higher on the North Fork. Diatom collections were made from multiple sites on each branch. Identifications to genera and counts of 200 were made. An identified list of the diatoms present was compiled for each site, and the differences are reported. This project has direct implications on management strategies for the uppermost Roanoke River Drainage.