Regulators of biotransformation are of particular interest in pharmacology and toxicology, determining in part the metabolism, disposition, and toxicity of chemicals. The nuclear receptor NR1I2 (pregnane X receptor, PXR) is a prominent xenosensor that regulates the expression of biotransformation enzymes governing elimination of many exogenous as well as endogenous compounds. Zebrafish (Danio rerio) has only one gene locus for pxr, but different genetic variants have been identified in zebrafish. However, the prevalence and significance of these variants are unknown. We hypothesize that sequence variation occurring in the Pxr gene of zebrafish may affect the action and fate of many chemicals in this species, a key model organism in various fields of research, including environmental toxicology. Here, we examine variation in Pxr sequences from four different strains of zebrafish and assess the responses of each Pxr to clotrimazole and butyl-4-aminobenzoate. The Pxr variants differed in both their ability to bind these structurally different ligands and to regulate reporter gene expression in vitro. We infer that the observed sequence variations in zebrafish Pxrs likely affect the response to putative Pxr agonists in vivo and potentially cause strain-specific biotransformation of xenobiotics in zebrafish. Thus, the choice of zebrafish strain could affect the outcome of downstream toxicological studies.