Antimicrobial resistance is an emerging threat. The Chesapeake Bay and upper watershed were previously found to harbor AR in multiple bacteria during the summer months. During the winter of 2013, we conducted a pilot study to survey bacterial diversity and resistance in isolates from the Chesapeake Bay and upper watershed. Water samples were collected from nine sites on the Chesapeake Bay and upper watershed. All bacterial isolates were identified to the species level using MALDI-ToF MS. Antimicrobial susceptibility testing was performed for all Gram-negative isolates using Etest and 14 antibiotics. A total of 35 bacterial isolates were recovered from all sites, including 8 Gram-positive and 27 Gram-negative organisms. Resistance to chloramphenicol and imipenem exists in the Chesapeake Bay and upper watershed. Further studies are needed to better understand the antibiotic resistance identified in this study and potential sources of antibiotic exposure and transmission.