Laboratory animals tend to be more inbred and less genetically diverse than wild populations, and thus may differ in their susceptibility to chemical stressors. We tested this hypothesis by comparing the responses of related inbred (theoretical inbreeding F(IT) = n + 0.25) and outbred (F(IT) = n) zebrafish (Danio rerio) WIK/Wild family lines to an endocrine disrupting chemical, clotrimazole. Exposure of inbred and outbred zebrafish to 2.9 μg clotrimazole/L had no effect on survival, growth, or gonadal development. Exposure of both lines to 43.7 μg clotrimazole/L led to male-biased sex ratios compared with controls (87% versus 55% and 92% vs 64%, for inbred and outbred males, respectively), advanced germ cell development, and reduced plasma 11-ketotestosterone concentrations in males. However, outbred males (but not inbred males) developed testis that were more than twice the weight of controls, which corresponded with a proliferation of Leydig cells and maintenance of the expression (rather than down-regulation occurring in inbreds) of gonadal aromatase (cyp19a1a) and insulin-like growth factor (igf1). Our results illustrate that the effects of an endocrine disrupting chemical (clotrimazole) on some end points (here testis development) can differ between inbred and outbred zebrafish. This highlights the need for reporting pedigree/genetic information and consistency in the responses of laboratory animals (e.g., by using model compounds as positive controls).