AbstractFour methicillin-resistantStaphylococcus sciuri(MRSS) strains isolated from stranded dogs showed trimethoprim (TMP) resistance, while all staphylococcal TMP resistant dihydrofolate reductase genes (dfr) were negative. An in-depth whole-genome-sequencing approach on strain C2865 was followed forresistomeandmobilomeprofiling, and for comparative genomics withS. sciurigroup available genomes. Lack of species host tropism was observed, with MRSS C2865 placed at a separate sub-branch withinS. sciurispecies, close to the average nucleotide identity to be considered a different species (95-96%).S. sciuriproved a pronounced accessory genome (73% of genes), while MRSS C2865 distinctively harboured the highest total gene number and highest number of unique genes, with 75% associated to the recognisedmobilome. A novel multidrug resistance mosaic plasmid (pUR2865-34) with several adaptive, mobilization (oriTmimic) and segregational stability (Type Ibparsystem) traits and two small single resistance plasmids were identified. Plasmid pUR2865-34 enclosed a novel staphylococcal TMP resistance gene, nameddfrE, which shared highest identity withdfrof soil-relatedPaenibacillus anaericanus(68%). DfrE conferred high-level TMP resistance inS. aureusandEscherichia coli. Database searches revealed thatdfrEwas formerly denoted (dfr_like) in anExiguobacteriumspp. from a fish-farm sediment and that was present but unnoticed in several staphylococcal and onemacrococcal genomes with different epidemiological backgrounds. Novel chromosomal site-specific mobile islands with resourceful traits were identified, including a multidrug-resistant SCCmeccassette lacking cassette chromosome recombinase (Ccr) genes, a staphylococcal pathogenicity island of the SaPI4 family, and three unrelatedsiphoviridaeprophages, two of which enclosed recombinases with the conserved Ccr-motif. We reveal a novel staphylococcal TMP resistancedfrEgene already present in diverse bacterial backgrounds. We confirm the ubiquity, high genome plasticity and low host tropism ofS. sciurihighlighting its role as a resourceful reservoir for evolutionary novel features contributing to its extraordinary versatility and adaptability.Author summaryStaphylococcusspp. are ubiquitous bacteria present in diverse ecological niches, including humans, animals and the environment. They are clinically relevant opportunistic pathogens and are notorious for their ability to acquire antimicrobial resistance (AMR) and virulence properties, resulting in a significant impact for Public Health. Mobile genetic elements (MGEs) play a central role in this adaptation process and are a means to transfer genetic information across bacterial species.Staphylococcus sciurirepresents one of the most ancestral species in the genus and has been suggested a reservoir for AMR genes. Here, following a refined whole genome sequencing approach we determined the entire genome of an animal and environment-associated multidrug resistant (MDR)S. sciuristrain uncovering a novel acquired staphylococcal TMP resistance gene already spread among different bacterial species from different epidemiological backgrounds. We also reveal several additional MGEs, including a novel MDR mobilizable plasmid that encloses several adaptive and stabilization features, and novel mobilizable chromosomal islands with resourceful traits, including three unrelated prophages. Together with comparative genomics, we confirm the ubiquity, high intraspecies heterogenicity, genome plasticity and low host tropism of this species, highlighting its role as resourceful reservoir for evolutionary novel features contributing to its extraordinary versatility and adaptability.