The common practice of using therapeutic antibiotics in livestock farming is a worldwide phenomenon. Over the last decade, there has been a growing concern of antibiotics entering the environment via animal manure. Similar studies have focused on the occurrence and biological effects of antibiotics in land‐applied animal feedlots; however, limited research has been conducted on the occurrence and persistence of antibiotics in animal feedlots. Therefore, the objective of this study was to evaluate antibiotic persistence, fate, and transport in surface water runoff and feedlot sediment in feedlot pens with livestock either receiving or not receiving antibiotic treatments through injection and feed. The two antibiotics (tylosin and monensin) added to animal feed were observed to persist in the soil environment for more than 30 days along with injected florfenicol. Monensin (5.6X higher) and tylosin (20X higher) were significantly higher in livestock pens receiving antibiotics compared livestock pens not receiving the antibiotics. Further, rainfall was observed to significantly impact soil surface concentrations of florfenicol. Other antibiotics administrated by injection were not observed to statistical increase in concentrations in runoff or feedlot sediment. Findings emphasize antibiotics administered in feedlots have the potential to persistent and remain in feedlot sediment and runoff, particularly in instances of regular administration in feed.This article is protected by copyright. All rights reserved