Abstract: Antimicrobial resistance poses a major threat to human and wildlife health. Although migratory waterbirds can carry antibiotic resistance genes (ARGs), cross-species ARG sharing and horizontal gene transfer (HGT) risks remain poorly understood under mixed-species overwintering conditions. We combined metagenomic sequencing with analyses of host phylogenetic relationships and species divergence times to characterize the gut resistomes of four mixed-species overwintering waterbirds. We identified 844 ARG subtypes across 24 resistance classes. β-lactam resistance genes exhibited high subtype richness, whereas multidrug resistance genes dominated total ARG abundance. ARG composition differed among host species but did not fully correspond to host phylogeny or divergence history. Among the 225 ARG subtypes shared across all four species, 39 core subtypes accounted for 50.4–61.7% of total ARG abundance in each species. We further identified 39 high-risk ARG subtypes, comprising 28 Rank I and 11 Rank II ARGs across 10 antibiotic classes. Across all samples, we detected 127 mobile genetic element (MGE) subtypes, of which insertion sequences and transposase-associated elements predominated, together accounting for 86.8–97.7% of total MGE abundance in each host species. Colocalization of a subset of ARGs (e.g., sul1 and catB3) with MGE-associated markers (e.g., intI1 and tnpA) suggested the potential for MGE-mediated horizontal transfer of these ARGs. Potential pathogens, including Escherichia coli and Salmonella enterica, were also identified as hosts of potentially mobile ARGs. Overall, this study reveals extensive cross-species ARG sharing and potential HGT risk in mixed-species overwintering waterbirds, highlighting their potential role in mediating ARG dissemination across host species.

Key words: antibiotic resistance genes, horizontal gene transfer, metagenomic sequencing, overwintering waterbirds