Simple SummaryThe aim of this study was to investigate the invasive speciesLampropeltis californiae(California kingsnake) as a reservoir ofSalmonellaand its ability to spread different clones of the bacterium with zoonotic potential into the environment, as well as study its antimicrobial resistance patterns in Gran Canaria (Spain). The main results showed that a high diversity ofSalmonellasubsp.salamaestrains circulate in Gran Canaria with a high prevalence of resistance shown for antimicrobials of public health importance, as summarised in the European Decision 2013/652/EU.AbstractThe increase in the reptile population has led to a rise in the number of zoonotic infections due to close contact with reptiles, with reptile-associated salmonellosis being particularly relevant. California kingsnake invasion not only threatens the endemic reptile population of the island of Gran Canaria (Spain) but also poses serious public health problems by spreading zoonotic pathogens and their antimicrobial resistance (AMR) to the environment. Thus, the aim of this study was to assess the occurrence, genetic diversity, and AMR amongSalmonellaspp. strains isolated from California kingsnakes in Gran Canaria Island (Spain). Of 73 invasive individuals captured, 20.5% carriedSalmonellaspp., belonging to different subspecies and serovars, with subsp.salamaeas the most abundant. Pulsed-field electrophoresis showed high genetic diversity among subsp.salamaeisolates, and among these, 73.3% showed resistance to at least one of the antimicrobials tested. In conclusion, the present study revealed the importance of wild invasive California kingsnakes as reservoirs of drug-resistantSalmonellaspp. that could pose a direct threat to livestock and humans. Identification of drug-resistantSalmonellastrains in wildlife provides valuable information on potential routes of transmission that involve risks to public and animal health.