Native fishes of the Great Plains are at risk of decline due to disturbances to physical habitat caused by changes in land and water use, as well as shifts in species assemblages driven by the invasion of introduced species with the loss of natives. We used historical and current fish assemblage data in conjunction with current habitat information to assess these influences on an endemic Great Plains stream fish, the plains topminnow (Fundulus sciadicus). Of the 31 sites where the plains topminnow occurred historically (1939–1940), it was found in only seven of those sites in 2003–2005. Our results demonstrate a shift in fish assemblage over time that coincides with the loss of plains topminnow. Changes in fish assemblages were characterized by increases in occurrence of exotic, invasive and generalist species with declines in occurrences of native fishes. An information theoretic approach was used to evaluate candidate models of current fish assemblage and physical/chemical habitat on the presence of the plains topminnow. Candidate models that included both instream habitat (e.g., vegetation coverage, undercut banks) and the native fish species assemblage are important to predicting presence of the plains topminnow within its historic range. Conservation of Great Plains fishes including the plains topminnow will need a combination of habitat protection and enhancement.