Mechanisms of the displacement of native fish by nonnative fish can include agonistic behaviors that push native fish species out of their preferred habitat, including their thermal optima. To examine these interactions, we built an experimental thermal preference chamber to evaluate: (1) the thermal preference of native, glacial relict northern redbelly dace Chrosomus eos; (2) if the thermal preference and movement changed in the presence of the invasive western mosquitofish Gambusia affinis; and (3) the direction of agonistic interactions. We hypothesized that G. affinis would express agonistic behavior toward C. eos, because G. affinis is widely recognized as an aggressive invader. Given the temperature range of the experimental chamber, i.e., 20–30 °C, C. eos selected an average of 24.3 °C as its thermal preference. After G. affinis’ introduction, the thermal preference of C. eos increased by 1.7 °C and the movement, given by distance (cm) travelled, increased by 21%. Contrary to our prediction, more agonistic interactions were observed in C. eos toward G. affinis. These results indicate that agonistic behavior of G. affinis toward native fish species may be species- and condition-specific, and may not always be the primary mechanism of native species’ displacement. Biological invasions are a global issue and altered thermal regimes are expected to continue. This study provided the novel approach using of a thermally heterogeneous thermal chamber to examine thermal preferences and aggressive interactions between a native and an invasive species. Future research should examine other life history traits that may be conveying the competitive advantage to G. affinis.