Antibiotics are commonly used in agriculture and industry and their discharge is commonly seen in rivers, like the Buffalo River. To study the effect of urban pollution on the antibiotic resistance in Aeromonas, 229 Aeromonas isolates were collected from fish tissues as well as sediment and water samples collected from the Buffalo River and a non-urban site (Cazenovia Creek). Seven different Aeromonas taxa were identified using biochemical tests. All Aeromonas isolates were tested for their resistance to six different antibiotics (cephalothin, cefoxitin, ceftriaxone, nalidixic acid, piperacillin, and tetracycline). This study can be used to guide future studies in antibiotic resistance from the Buffalo River watershed.