Stenotrophomonas maltophilia, an aerobic, gram-negative bacillus, displays multiple resistances to common antibiotics. Two indigenous -lactamases encoded by chromosomal genes, L1 and L2, protect S. maltophilia from attack by -lactam antibiotics. Heterogeneity of L2 gene is known to be highly prominent in S. maltophilia. In this study, the L2 sequences is obtained from PCR and further sequenced from the 22 clinical isolates. A series of bioinformatics analysis about L2 is performed, including multiple alignment, allelic variation analysis, phylogenetic analysis, and characteristics prediction. A phylogenetic tree is derived based on the DNA sequences of L2 genes from the 22 clinical isolated and seven L2 genes in the GenBank databases. Nine phylogenetic groups are identified and four L2 phylogenetic groups are reported for the first time. To understand the relationship between genotype and phenotype of L2, the L2 gene is cloned into vector and the resultant recombinant plasmid is transformed into E. coli cell. The expressed L2 activity of recombinant E. coli cell is detected by susceptibility test. No detectable -lactamase activity is expressed in all test strains. AmpR gene, located in the upstream of L2 gene is reported as a transcriptional regulator, affecting the expression of L2 gene. The DNA fragments containing ampR and L2 are got by PCR from four clinical isolates. Similar bioinformatics analysis is carried on. To further investigate the effect of AmpR on the -lactamase activity expression of L2, the -lactamase activity of recombinant E. coli cell containing the constructed ampR-L2-plasmid is evaluated. The -lactamase activities of strains E. coli DH5(pOKM2AmpR-L2) and E. coli DH5(pOKATCC13637AmpR-L2) are higher than that of E. coli DH5(pOK12).