In this study, we examine how the Guri catfish Genidens genidens uses estuarine and freshwater habitats along the largest South American coastal lagoon, through the chemical analysis of otoliths and microscopic analysis of gonads. Chemical composition (Sr:Ca) of otolith edges allowed distinguishing between individuals who used the estuarine or freshwater compartments of the lagoon. The analysis of core-to-edge chemical profiles of each individual otolith revealed that the population may present two different patterns of habitat use along the lagoon. The ‘type 1’ pattern (89.5%) includes fish who appear to have been born in estuarine waters, whereas ‘type 2’ (9.5%) includes those fish born in fresh water. Nevertheless, juveniles from both patterns appear to migrate to estuarine waters. The gonad analysis shows G. genidens may reproduce in fresh water, as nearly 57% of all sampled fish were found to spawn in the freshwater portion of the lagoon. Also, the otolith core of many adult fish presented freshwater signatures, thus suggesting consistent fresh water use during early life. Our findings based on otolith and gonadal analyses challenge the previous classification of G. genidens as an estuarine resident. Rather, our results allow the suggestion that this species should be placed in the ‘estuarine and fresh water’ guild, which includes both fish completing their life cycles within the estuary and fish who consistently use freshwater habitats.