Fish otolith and water chemistry were assessed in the Grand Canyon reach of the Colorado River and its tributaries. Aqueous strontium and selenium (in ratio to calcium) and carbon stable isotopic ratios were identified as markers with excellent potential to track the provenance and movements of the endangered humpback chub Gila cypha. Although otolith δ13C and Sr/Ca varied proportionately to water chemistry and provided a framework for detailed study of humpback chub movements, otolith Se/Ca showed ambiguous tracking of known water chemistries. As an application, we document the natal source and movement dynamics of n = 10 humpback chub and compare these findings from otolith microchemistry with the current paradigm of humpback chub spawning ecology. We found that seven of ten fish follow the current early life history paradigm and were spawned in the Little Colorado River and subsequently emigrated to the main stem Colorado River as juveniles. However, the otolith markers of three fish suggest an alternative early life trajectory with unknown provenance. Age and growth analyses demonstrate seasonally higher growth rates in the warmer Little Colorado River compared with the Colorado River. Combining natural markers with age and growth reconstructions provides a powerful tool for assessing the habitat use and success of humpback chub in the Grand Canyon. Copyright © 2012 John Wiley & Sons, Ltd.