Two hundred clinical isolates of Stenotrophomonas maltophilia were examined for the presence of class 1 integron and for the susceptibility to 12 different antimicrobials and detergents. The prevalence of class 1 integron in S. maltophilia isolates was 11%. The class 1 integron-positive isolates exhibited a higher resistance to kanamycin, tobramycin, and trimethoprim–sulfamethoxazole (SXT) than the class 1 integron-negative ones. Polymerase chain reaction (PCR), amplifying the variable region of the class 1 integron, showed the existence of six different amplicon sizes, indicating that there are at least six different class 1 integrons distributed in the 23 class 1 integron-positive isolates. Sequence analysis of six representative PCR amplicons revealed that qacK , aac(6′)-Ib′ , qacK-aac(6′)-Ib , qacK-aac(6′)-Ib-aac(6′)-Ib , and qacL-aadB-cmlA-aadA2 were identified in the 550-, 800-, 1,200-, 1,800, and 3,600-bp amplicons, respectively. The sequence analysis of the 150-bp PCR amplicon demonstrated no additional resistance-associated genes except the basic genetic elements of class 1 integron. The impact of class 1 integron acquisition on the antimicrobials susceptibility was assayed by isogenic integron deletion mutant construction and the susceptibility test. The most significant contribution of the class 1 integron acquisition to S. maltophilia is the increased resistance to SXT.