Biotic communities and environmental conditions can be highly variable between natural ecosystems. The variability of natural assemblages should be considered in the interpretation of any ecological study when samples are either spatially or temporally distributed. Little is known about biotic variability in the Santa Ana River Basin. In this report, the lotic community and habitat assessment data from ecological studies done as part of the U.S. Geological Survey's National Water-Quality Assessment (NAWQA) program are used for a preliminary assessment of variability in the Santa Ana Basin. Habitat was assessed, and benthic algae, benthic macroinvertebrate, and fish samples were collected at four sites during 1999-2001. Three of these sites were sampled all three years. One of these sites is located in the San Bernardino Mountains, and the other two sites are located in the alluvial basin. Analysis of variance determined that the three sites with multiyear data were significantly different for 41 benthic algae metrics and 65 macroinvertebrate metrics and fish communities. Coefficients of variation (CVs) were calculated for the habitat measurements, metrics of benthic algae, and macroinvertebrate data as measures of variability. Annual variability of habitat data was generally greater at the mountain site than at the basin sites. The mountain site had higher CVs for water temperature, depth, velocity, canopy angle, streambed substrate, and most water-quality variables. In general, CVs of most benthic algae metrics calculated from the richest-targeted habitat (RTH) samples were greater at the mountain site. In contrast, CVs of most benthic algae metrics calculated from depositional-targeted habitat (DTH) samples were lower at the mountain site. In general, CVs of macroinvertebrate metrics calculated from qualitative multihabitat (QMH) samples were lower at the mountain site. In contrast, CVs of many metrics calculated from RTH samples were greater at the mountain site than at one of the basin sites. Fish communities were more variable at the basin sites because more species were present at these sites. Annual variability of benthic algae metrics was related to annual variability in habitat variables. The CVs of benthic algae metrics related to the most CVs of habitat variables included QMH taxon richness, the RTH percentage richness, RTH abundance of tolerant taxa, RTH percentage richness of halophilic diatoms, RTH percentage abundance of sestonic diatoms, DTH percentage richness of nitrogen heterotrophic diatoms, and DTH pollution tolerance index. The CVs of macroinvertebrate metrics related to the most CVs of habitat variables included the RTH trichoptera, RTH EPT, RTH scraper richness, RTH nonchironomid dipteran abundance (in percent), and RTH EPA (U.S. Environmental Protection Agency) tolerance, which is based on abundance. Many of the CVs of habitat variables related to CVs of macroinvertebrate metrics were the same habitat variables that were related to the CVs of benthic algae metrics. On the basis of these results, annual variability may have a role in the relationship of benthic algae and macroinvertebrates assemblages with habitat and water quality in the Santa Ana Basin. This report provides valuable baseline data on the variability of biological communities in the Santa Ana Basin.