The Trichechus manatus latirostris (Florida manatee) is known to seasonally range into the northern Gulf of Mexico, but evidence of mating behavior in Mississippi has not previously been documented. On 24 September 2024, the first known instance of active manatee mating behavior in Mississippi waters was observed using an uncrewed aerial system (UAS) at the Grand Bay National Estuarine Research Reserve. Behavioral analyses, including all-occurrence and continuous sampling methods, revealed high frequencies of mounting interactions characteristic of manatee reproductive behavior. The focal group consisted of three individuals, with one identified female (Manatee B) and presumed male (Manatee A) engaging repeatedly in mounting behaviors. This novel mating behavior differs from the typical “mating herd” of manatees consisting of multiple males with a single female. These findings suggest that Mississippi coastal habitats may support not only seasonal foraging but also reproductive activity, indicating a potential range expansion and reproductive plasticity of the species. This observation underscores the need for enhanced monitoring and conservation efforts in northern Gulf habitats to account for emerging manatee behaviors and potential establishment of reproductive use areas beyond Florida.