Evolutionary changes in steroid hormone receptor sequences have led to species variation in the ligands of these receptors and hormone function (Eick et al., 2012). This study examined how variation in responsiveness of a sexually dimorphic communication signal (the electric organ discharge of electric fish) to androgens might be related to evolutionary changes in the sequence of the androgen receptor (AR). Knifefish communicate with continuously produced electric organ discharges (EODs). In many species, EOD frequency (EODf) differ between males and females. Sex differences in EODf are regulated by effects of steroid hormones in the hindbrain pacemaker nucleus (Pn). Two species of knifefish (A. albifrons and A. leptorhynchus) differ substantially in the efficacy of different androgens on EODf. 11-ketotestosterone (11-KT) robustly masculinizes EODf in both species (Schaefer and Zakon, 1996). However, 5-α dihydrotestosterone (5-α DHT) masculinizes EODf in A. albifrons, but does not affect EODf in A. leptorhynchus (Meyer et al., 1987). Two different AR genes (ARα and ARβ) were expressed. ARα had the highest expression in the Pn of both species. ARα was less conserved between A. albifrons and A. leptorhynchus than ARβ of these two species. When knifefish ARs were compared across species, ARα of A. leptorhynchus had more changes than other knifefish in key regions of the ligand binding domain. These “key regions” are residues in humans that directly interact with 5-αDHT (Ogino et al., 2015). These specific residues also tend to be highly conserved in AR of teleost fish. Overall, our data suggests that evolution of AR gene sequence change androgen selectivity in ways that could impact hormonal regulation of behavior.