It has been recently shown that Stenotrophomonas maltophilia contains a chromosomally encoded qnr gene (Smqnr) which confers low-level resistance to quinolones upon its expression in a heterologous host. However, the role of this chromosomally encoded Qnr determinant in the susceptibility of its original host to quinolones remains unknown, while plasmid-encoded Qnr determinants contributing to quinolone resistance via horizontal gene transfer have been well studied. Here, we analyzed the role of SmQnr in the intrinsic resistance of S. maltophilia to quinolones by comparing the susceptibility of a markerless Smqnr deletion mutant with that of its isogenic wild-type strain. Deletion of Smqnr rendered increased susceptibility to quinolones, demonstrating SmQnr's contribution to intrinsic resistance. Complementation with Smqnr restored quinolone resistance in the mutant, and overexpression of Smqnr (via plasmid encoding) further increased resistance, consistent with a gene dosage effect confirmed by real-time RT-PCR showing higher Smqnr expression in plasmid-encoded strains. This is the first demonstration of a role for chromosomally encoded Qnr determinants in intrinsic bacterial resistance to quinolones. Additionally, Smqnr overexpression might also be involved in acquired resistance in S. maltophilia.