Natural waters are contaminated with trace concentrations of antibiotics, most frequently sulfamethoxazole (SMX). Juvenile rainbow trout (Oncorhynchus mykiss) were exposed for six months to sub-inhibitory concentrations of SMX. In two independent trials, intestinal microbiota developed resistance to a high concentration of SMX (1,000,000 _g/L) after exposure to concentrations 100,000-fold less than the minimum inhibitory concentration. Highly resistant isolates did not contain sul1, a common sulfonamide resistance gene. Strikingly, of the isolates susceptible to 1,000,000 ug/L SMX, exposure to as low as 0.3 _g/L SMX, resulted in a prevalence of sul1 of over 89% compared to 14% in the no antibiotic group. This result suggests that sul1 may provide a fitness benefit at low levels of exposure to SMX. This study demonstrates a reproducible emergence of resistance to SMX and promotion of the sul1 antibiotic resistance gene with chronic exposure to SMX at concentrations as low as those found environmentally.