Karst aquifers provide a significant source of drinking water around the world; however, they are prone to bacterial contamination. We investigated the prevalence, serotypes, and antimicrobial resistance genes (ARGs) of Salmonella in urban karst groundwater and groundwater‐fed surface waters. Weekly water samples (n = 443) were collected from nine groundwater sites and the Barren River in Bowling Green, KY. Additionally, cross‐sectional samples were collected from 45 sites, including groundwaters and two rivers, in Tampa, FL. Salmonella was detected in 14.5% of Bowling Green samples resulting in 176 isolates representing 18 serotypes. Two (4.4%) of the Tampa samples were positive yielding eight isolates representing two serotypes. In Bowling Green, Salmonella prevalence varied by sampling site, week, month, and season, with the highest prevalence in surface water in the month of November and during summer. Salmonella Newport was the most common serotype detected. Nine of the serotypes detected in the study were among the top 20 serotypes commonly associated with human infections in the United States. Isolates were clustered with human clinical isolates, or isolates obtained from food animals, suggesting the importance of humans and agricultural practices as a source of water contamination. ARGs were detected in 12.5% (n = 176) of the Bowling Green isolates; overall, 81.8% (n = 22) of these were multidrug‐resistant (resistance to ≥3 antimicrobial classes). Notably, the four Salmonella Agona isolates were resistant to four antimicrobial classes, mercury, and quaternary ammonium compounds. Seasonality and the widespread occurrence of Salmonella both in the groundwater and groundwater‐fed surface waters is a potential risk to public health.