The Houston Ship Channel (HSC) in Houston, Texas is an aquatic environment with a long history of contamination, including polychlorinated dibenzo-p-dioxins (PCDD), polychlorinated dibenzofurans (PCDF), polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), and heavy metals. Populations of Gulf killifish (Fundulus grandis) from the HSC have adapted to resist developmental cardiac deformities caused by dioxin-like compounds (DLCs). Contaminants in the HSC have acted as a strong selective pressure on resident Gulf killifish populations. Rapid adaptation can lead to fitness costs, some as a direct result of the mechanisms involved in the adaptive process, whereas other adaptations may be more general. To explore potential fitness costs, we evaluated two Gulf killifish populations with documented resistance to DLC-induced cardiac teratogenesis (Patrick Bayou and Vince Bayou), and one previously characterized reference population (Gangs Bayou). We also characterized a previously unstudied population from Galveston Bay as an additional reference population (Smith Point). We tested the sensitivity of F1 larvae from these four populations to two classes of pesticides (pyrethroid (permethrin) and carbamate (carbaryl)) and two model pro-oxidants (tert-butyl hydroquinone (tBHQ) and tert -butyl hydroperoxide (tBOOH)). In addition, we explored their responses to hypoxia and measured resting metabolic rates (<img height="36" border="0" style="vertical-align:bottom" width="46" alt="View the MathML source" title="View the MathML source" src="http://origin-ars.els-cdn.com/content/image/1-s2.0-S0166445X16300820-si1.gif">M02⋅). Both adapted populations were cross-resistant to the toxicity of carbaryl and both pro-oxidants tested. There were no population differences in sensitivity to permethrin. On the other hand, one reference population (Gangs Bayou) was less sensitive to hypoxia, and maintained a lower <img height="36" border="0" style="vertical-align:bottom" width="46" alt="View the MathML source" title="View the MathML source" src="http://origin-ars.els-cdn.com/content/image/1-s2.0-S0166445X16300820-si1.gif">M02⋅. However, there were no differences in hypoxia tolerance or resting metabolic rate between the second reference and the two adapted populations. This investigation emphasizes the importance of including multiple reference populations to clearly link fitness costs or cross-resistance to pollution adaptation, rather than to unrelated environmental or ecological differences. When compared to previous literature on adapted populations of F. heteroclitus, we see a mixture of similarities and differences, suggesting that F. grandis adapted phenotypes likely involve multiple mechanisms, which may not be completely consistent among adapted populations.