This study identifies and characterizes a novel antimicrobial resistance-associated megaplasmid in Pseudomonas sp. isolated from diseased Nile tilapia (Oreochromis niloticus) in Mexico. Aquaculture’s growth has heightened concerns over antimicrobial resistance (AMR) due to the widespread prophylactic use of antibiotics to manage bacterial infections, which contributes to AMR in pathogens like Pseudomonas spp., an opportunistic agent of fish disease. A bacterial isolate from symptomatic tilapia was identified as closely related to Pseudomonas soli through 16S rRNA gene sequencing. Whole-genome sequencing revealed a 362,580 bp megaplasmid encoding 984 genes, including multidrug resistance elements, confirmed by PCR targeting key genes (repA and virB4/traC) (Figure 1). The megaplasmid exhibited high homology to known resistance plasmids, underscoring the potential for horizontal gene transfer in aquaculture environments. This first identification of such a megaplasmid in tilapia pathogens highlights the urgent need for sustainable antibiotic practices in aquaculture to mitigate AMR’s spread. Our findings contribute to understanding the genomic basis of AMR in Pseudomonas spp. and offer insights for developing management strategies to safeguard fish health and food security.