Stenotrophomonas maltophilia is known to produce at least two chromosomal-mediated inducible β-lactamases, L1 and L2. Gene L2, which encodes a class A β-lactamase, and the adjacent ampR gene form an ampR-class A β-lactamase module. L1 belongs to the class B β-lactamase and has no neighbor ampR-like regulatory gene. In this study, the ampR-L2 module from S. maltophilia KH was compared with ampR-β-lactamase modules from several microorganisms with respect to the AmpR and β-lactamase proteins and the intergenic (IG) region. S. maltophilia and Xanthomonas campestris showed the most closely phylogenetic relationship among the microorganisms considered. The regulatory role of AmpR towards L1 and L2 was further analyzed. In the absence of an inducer, AmpR acted as an activator for L1 expression and as a repressor for L2 expression, whereas AmpR was an activator for both genes in an induced state. In addition, inducibility of L1 and L2 genes depended on the presence of AmpR. The ampR transcript was weakly and constitutively expressed, but was not autoregulated.