Naturally occurring chemical markers in otoliths offer a potential but untested means to identify source environment for fishes in the upper Illinois River system and Lake Michigan, including individuals that may breach or circumvent electrical barriers in the Chicago Sanitary and Ship Canal or be transferred via bait buckets between these formerly isolated drainages. The objectives of this study were to determine whether water and fish otolith stable isotopic and elemental compositions differ among Lake Michigan, the upper Illinois River, and three tributaries of the upper Illinois River (Fox, Des Plaines and DuPage Rivers) and to determine whether otolith isotopic and elemental signatures could be used to identify the water body from which individual fish were collected. Water and fish otolith samples were obtained from each site during 2007 and analyzed for δ¹⁸O and a suite of trace element concentrations; otoliths also were analyzed for δ¹³C. Otolith δ¹³C values for Lake Michigan fish were distinct from individuals collected in the Illinois River and tributaries. Fish collected in the Fox and Des Plaines Rivers could be distinguished from one another and from fish captured in the Illinois and DuPage Rivers using otolith Sr:Ca and Ba:Ca ratios. Otolith isotopic and elemental compositions may enable identification of source environment for fishes that move or are transferred between the Illinois River drainage and Lake Michigan; however, temporal variation in otolith chemical signatures should be assessed.