Our goal was to identify habitat, fish species, and fish assemblages associated with the occurrence of Topeka Shiners Notropis topeka in stream and off-channel habitat (OCH) of west-central Iowa. Fish assemblages and habitat characteristics were estimated in 67 stream and 27OCHsites during 2010–2011. Topeka Shiners were sampled in 52% of OCH sites, but in only 9% of stream sites, which supports the hypothesis that OCH is an important component of their life history. Fish assemblages containing Topeka Shiners were different from those that did not contain Topeka Shiners in OCH sites, but this was not evident in stream sites. Results from logistic regression models suggested that Topeka Shiner presence was associated with increased submerged vegetation and abundance of Fathead Minnow Pimephales promelas. Contrary to the findings of other studies, the abundance of large piscivorous fishes was not associated with the occurrence of Topeka Shiners. Our results provide new information about the biology and life history of the Topeka Shiner that will guide habitat restoration and other recovery efforts.