The use of fatty acid (FA) analysis has become an important tool in recent years to investigate a broad spectrum of questions in fisheries and aquatic ecology. One of these applications has been the use of FA profiles as biomarkers for indicating diet and recent feeding history of fishes and other aquatic consumers. Differences in forage FA profiles and spatial differences in food web structure within aquatic systems may lead to corresponding spatial patterns of FA profiles in consumers, thereby facilitating the potential applicability of FA biomarkers as indicators of habitat use and origin for mobile consumers such as fishes. However, little information is available regarding differences in FA profiles among habitat types in river-floodplain ecosystems and the time-scale over which fish FA profiles change when fish move between habitats that differ in the FA profiles of prey resources. The objectives of this study were to test whether the tissue FA profiles of channel catfish (Ictalurus punctatus) differed among three reaches of the lower Kaskaskia River and its floodplain lakes, to determine the time-course of tissue FA profile turnover when fish are relocated to a new habitat type, and to compare FA profiles among muscle, liver, and adipose fin tissues in channel catfish. Fatty acid profiles of channel catfish were significantly different among sites, especially between upper and lower river sites, and between river channel and oxbow lake sites, suggesting differing energy sources among habitats and river reaches in the Kaskaskia system. More specifically, there was a significant increase in the essential FAs, 18:2n-6 and 18:3n-3, in channel catfish with increasing distance downstream, which could reflect an increase in river-floodplain connectivity at downstream sites. Channel catfish transplanted from the Kaskaskia River to ponds at Southern Illinois University Carbondale showed a significant linear decrease in n-3 and medium-chain polyunsaturated FAs (MC-PUFA), and a significant linear increase in monounsaturates (MUFA) with longer residence time in a lentic environment. Liver tissue exhibited the fastest turnover rate of the three tissue types (< 2 weeks), with muscle and adipose fin tissue both displaying similar, longer turnover times (approximately 10 weeks). Results of this study support the use of FA profiles as indicators of energy sources for fishes in large river-floodplain ecosystems, including the potential for indentifying habitat-specific (river channel vs. floodplain lake) or river reach-specific energy sources. Additionally, results of this study provide a timeline for FA turnover in channel catfish tissues when fish move among habitats with distinct FA signatures, which is crucial for detecting temporal shifts in use of habitat-specific energy sources by channel catfish. Results also suggest that adipose fin tissue samples may be a less invasive alternative to muscle tissue for analysis of FA profiles in channel catfish. Improved understanding of which habitats or river reaches provide trophic support for riverine fishes through use of FA profile analysis has potentially important implications for habitat conservation and rehabilitation in river-floodplain ecosystem.