Genome-based taxonomy reveals hidden diversity within the reorganized Stenotrophomonas genus and supports the proposal of a novel species

The genus Stenotrophomonas comprises globally distributed bacteria of increasing clinical relevance, characterized by intrinsic multidrug resistance and remarkable ecological versatility. Species-level identification within this group remains challenging as formal classifications in public databases continue to underestimate its true diversity. A recent genome-based reclassification proposed the division of the genus into five distinct genera: Allostenotrophomonas , Parastenotrophomonas , Pedostenotrophomonas , Pseudostenotrophomonas , and Stenotrophomonas , introducing further taxonomic uncertainty in an already complex and clinically important lineage. In this study we performed a comprehensive genome-based analysis of 146 genomes, previously classified as Stenotrophomonas , including 22 isolates recovered from hospital sink drains. Using average nucleotide identity (ANI), digital DNA–DNA hybridization (dDDH), and average amino acid identity (AAI), we identified 31 species-level clusters within the redefined Stenotrophomonas sensu stricto lineage, including sixteen potential new species. Our findings support the proposal of a novel species, Stenotrophomonas balearica sp. nov. (type strain GM_D5_149 T = CCUG 78226 T = CECT 31222 T ). Pan-genome analysis revealed an open pan-genome with a highly diverse accessory genome, reflecting the genus’s genetic plasticity and ecological adaptability. Putative resistance and virulence determinants were unevenly distributed across lineages, with enrichment within the S. maltophilia complex, reinforcing its clinical significance. Overall, our findings clarify species boundaries, reveal extensive cryptic diversity, and provide a genome-resolved framework for accurate taxonomic assignment within this increasingly relevant bacterial group. These results underscore the importance of genome-based systematics for improving diagnostic accuracy, epidemiological surveillance, and antimicrobial resistance monitoring.