Myroides odoratus and Myroides odoratimimus (formerly designated in a single species as Flavobacterium odoratum) are gram-negative aerobes and sources of nosocomial infections in humans. They have variable susceptibility to β-lactams and a decreased susceptibility to carbapenems. Using genomic DNAs of M. odoratus CIP 103105 and M. odoratimimus CIP 103073 reference strains, shotgun cloning of β-lactamase genes was performed, followed by protein expression in Escherichia coli. The deduced amino acid sequences of these β-lactamase genes revealed that TUS-1 and MUS-1 from M. odoratus CIP 103105 and M. odoratimimus CIP 103073, respectively, shared 73% amino acid identity. Mature proteins TUS-1 and MUS-1, with pI values of 7.8 and 5.2, respectively, had relative molecular masses of ca. 26 kDa. These β-lactamases are members of the subclass B1 of metallo-β-lactamases and are distantly related to other metalloenzymes, being most closely related to IND-1 from Chryseobacterium indologenes (42% amino acid identity). However, phylogenic analysis showed that TUS-1 and MUS-1 belong to the same phylogenic lineage of subclass B1 enzymes that groups the subclass B1 β-lactamases of Flavobacterium species. Kinetic parameters of purified β-lactamases TUS-1 and MUS-1 detailed their hydrolysis spectra, which encompass most β-lactams except aztreonam. β-Lactamases TUS-1 and MUS-1 were classified in functional subgroup 3a of metalloenzymes. This work further characterizes chromosome-encoded metalloenzymes from Flavobacteriaceae species that explain at least part of their intrinsic resistance to β-lactams.