To aid in assessing the extent of degradation in non-tidal streams, a multi-metric Index of Biotic Integrity (IBI) based on fish assemblages was developed for the Maryland Biological Stream Survey (MBSS). The MBSS is a probability-based, statewide monitoring program designed to assess the status of biological resources and to evaluate the effects of anthropogenic activities. We used data from more than 900 MBSS sites sampled in 1994-97 to refine and validate the IBI. Three distinct geographic strata (regions), corresponding with ecoregional and physiographic boundaries, were identified via cluster analysis and multivariate analysis of variance (MANOVA) as supporting distinctly different species groups. Reference conditions were based on minimally degraded sites that were sampled in 1994-97. We quantitatively evaluated the ability of various attributes of the fish assemblage (candidate metrics) to discriminate between these reference sites and sites known to be degraded, using statistical tests and classification efficiency. Formulations of the IBI were selected for each region based on high classification efficiency and broad representation of fish assemblage attributes.