Alligator Gar Atractosteus spatula are increasingly being stocked to restore populations, making the need to identify stocked individuals important for monitoring. Oxytetracycline (OTC) immersion allows for large numbers of fish to be marked simultaneously, thus eliminating the need to handle fish individually, but protocols for doing so have not been investigated fully for this species. In this study, we sought to identify dosages of OTC (concentration and duration of exposure) that would successfully mark juvenile Alligator Gar while minimizing mortality as a result of the marking procedures. Juvenile Alligator Gar (38 ± 4.4 mm [mean ± SE]) were collected from raceways at Tishomingo National Fish Hatchery 18–22 d after hatch and were marked during transport to the Oklahoma Fishery Research Laboratory. Ten individuals per treatment (360 total individuals) were randomly placed into one of three replicates containing one of four concentration × duration combinations of Pennox 343 OTC solution. Juvenile Alligator Gar were exposed to an OTC concentration of 0 (control), 500, 600, or 700 mg/L for a duration of 4, 5, or 6 h. Asteriscus, lapillus, and sagittal otoliths were examined for mark presence at 14 d postexposure by using fluorescent microscopy. The OTC concentration and duration both affected mean mark quality similarly among otolith types. Mortality increased with increasing OTC concentration, suggesting that a balance between concentration and duration is needed to achieve marking goals. Based on our findings, batch marking of Alligator Gar was successful at OTC concentrations from 500 to 700 mg/L for 4–6 h, although immersion at 500 mg/L for 6 h and 600 mg/L for 4–6 h produced the best balance between high mark quality and low associated mortality.