Antimicrobial-resistant bacteria (ARB) in aquatic environments pose problems to the environment and human health. Carbapenem is a strong antibiotic and is important because it has a great potency against infections by gram-positive and gram-negative bacteria. Carbapenem-resistant Enterobacteriaceae (CRE) are bacteria that belong to the Enterobacteriaceae family and can survive and grow against carbapenem antibiotics. Wastewater treatment plant (WWTP) effluents are the main source of those bacteria for surface waters in urban river settings. CRE genes have been shown to accumulate in different reservoirs in rivers such as periphyton, sediment, water, and invertebrates, while they move through food webs. Fish serve as bioindicators of CRE genes and can move these genes upstream and downstream while proliferating CRE genes in gut microbiota. The goal of this study is to investigate the environmental variables, particularly water quality factors, that influence CRE gene concentrations across different environmental matrices within the Ohio River Watershed. Samples were collected in the summer of 2021-2022 from major cities in Ohio at six different WWTPs. All samples were taken from upstream of outflow, outflow, and downstream of outflow from each site using both boat-mounted and backpack electrofishers. For fish gut, total phosphorus shows a significant positive correlation and temperature having a significant negative effect. In periphyton, total phosphorus remained a significant positive predictor, while dissolved oxygen exhibited a weak negative trend. In contrast to periphyton and fish gut, in the water matrix total phosphorus was negatively associated with CRE gene concentrations, while other water quality variables showed no significant correlations. These models show the role of total phosphorus as a key environmental driver influencing the dissemination of CRE genes across different matrices. This finding aligns with previous research indicating that phosphorus input from WWTPs can increase periphyton biomass, where CRE genes accumulate. As a result, periphyton feeders may be more likely to harbor higher concentrations of CRE.