The index of biotic integrity (IBI) has become a widely used tool for assessing the condition of stream fish communities and the overall biological status of streams. Because the location of a stream in a drainage network can influence the species richness offish communities and because species richness is an important component of the IBI, we examined the influence of stream spatial location on the IBI. We found that IBI scores for headwater streams in three Illinois drainage basins were significantly lower than those calculated for tributary streams of similar size connecting directly to larger streams. This difference in IBI was related to the increased species richness and to a greater number of sucker and darter species in tributaries that drain into larger, main‐channel streams. Because of the influence of tributary location on the IBI, expected values for headwater tributary streams should be developed independently from those developed for main‐channel tributary streams. Failure to do so can result in a substantial underestimation of the IBI of headwater tributary streams or an overestimation of main‐channel tributaries.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142315/1/tafs0635.pd