The contribution of human activities to environmental reservoirs of antibiotic resistance is poorly understood. The purpose of this study was to determine if oxytetracycline (OTC) use in aquaculture facilities increased the detection frequency (i.e., prevalence) of tetracycline resistance (tet(R)) genes relative to facilities with no recent OTC treatment. We used polymerase chain reaction to screen water and sediment from four noncommercial fish farms in northwestern Wisconsin for the presence of ten tet(R) determinants: tet(A), tet(B), tet(D), tet(E), tet(G), tet(M), tet(O), tet(Q), tet(S), and tet(W). Water from farms with recent OTC use had significantly higher tet(R) detection frequencies than did water from farms without recent OTC use, with prevalence in raceways and rearing ponds of farms with recent OTC use exceeding by more than twofold that of farms not using OTC. Effluent from all farms, regardless of treatment regime, had higher tet(R) detection frequencies than their corresponding influent for all genes, but the specific combinations of tet(R) genes detected in a sample were not different from their corresponding influent. Although OTC use was associated with the increased occurrence and diversity of tet(R) genes in water samples, it was not found to relate to tet(R) gene occurrence in sediment samples. Sediment samples from facilities with no recent OTC use had significantly higher frequencies of tet(R) gene detection than did samples from facilities with recent OTC use. All of the tet(R) genes were detected in both the medicated and nonmedicated feed samples analyzed in this study. These findings suggest that both OTC treatment in aquaculture facilities and the farms themselves may be sources of tet(R) gene introduction to the environment. To our knowledge, this is the first study to use genotypic and cultivation-independent methods to examine tet(R) gene occurrence associated with OTC use in aquaculture.