Effective water resources assessment and management requires quantitative information on the variability of ambient and biological conditions in aquatic communities. Although it is understood that natural systems are variable, robust estimates of long-term variation in community-based structure and function metrics are rare in U.S. waters. We used a multi-year, seasonally sampled dataset from multiple sites (n = 5-6) in four streams (Codorus Creek, PA; Leaf River, MS; McKenzie and Willamette Rivers, OR) to examine spatial and temporal variation in periphyton chlorophyll a, and fish and macroinvertebrate metrics commonly used in bioassessment programs. Within-site variation of macroinvertebrate metrics and benthic chlorophyll a concentration showed coefficient of variation ranging from 16 to 136%. Scale-specific variability patterns (stream-wide, season, site, and site-season patterns) in standardized biotic endpoints showed that within-site variability patterns extended across sites with variability greatest in chlorophyll a and lowest in Hilsenhoff's Biotic Index. Across streams, variance components models showed that variance attributed to the interaction of space and time and sample variance accounted for the majority of variation in macroinvertebrate metrics and chlorophyll a, while most variation in fish metrics was attributed to sample variance. Clear temporal patterns in measured endpoints were rare and not specific to any one stream or assemblage, while apparent shifts in metric variability related to point source discharges were seen only in McKenzie River macroinvertebrate metrics in the fall. Results from this study demonstrate the need to consider and understand spatial, seasonal, and longer term variability in the development of bioassessment programs and subsequent decisions.