Important natural environmental gradients, including the connectivity of off-channel aquatic habitats to the main-stem river, have been lost in many reaches of the upper Mississippi River system, and an understanding of the consequences of this isolation is lacking in regard to native fish communities. The objectives of this study were to describe patterns of fish species richness, evenness, and diversity among representative habitats and river reaches and to examine the relationship between fish species richness and habitat diversity. Each year (1994-1999) fish communities of main-channel borders (MCB), side channel borders (SCB), and contiguous backwater shorelines (BWS) were sampled using boat-mounted electrofishing, mini-fyke-nets, tyke nets, hoop nets, and seines at a standardized number of sites. A total of 0.65 million fish were collected, representing 106 species from upper Mississippi River Pools 4, 8, 13, and 26; the open (unimpounded) river reach; and the La Grange Reach of the Illinois River. Within pools, species richness based on rarefaction differed significantly among habitats and was highest in BWS and lowest in MCB (P < 0.0001). At the reach scale, Pools 4, 8, and 13 consistently had the highest species richness and Pool 26, the open-river reach, and the La Grange Reach were significantly lower (P < 0.0001). Species evenness and diversity indices showed similar trends. The relationship between native fish species richness and habitat diversity was highly significant (r(2) = 0.85; P = 0.0091). These results support efforts aimed at the conservation and enhancement of connected side channels and backwaters. Although constrained by dams, pools with high native species richness could serve as a relative reference. The remnants of natural riverine dynamics that remain in these reaches should be preserved and enhanced; conditions could be used to guide restoration activities in more degraded reaches.