Stenotrophomonas maltophilia is a multidrug-resistant organism that is emerging as an important opportunistic pathogen. Despite this, information on the epidemiology and characteristics of this bacterium, especially in Thailand, is rarely found. This study aimed to determine the demographic, genotypic, and phenotypic characteristics of S. maltophilia isolates from Maharaj Nakorn Chiang Mai Hospital, Thailand. A total of 200 S. maltophilia isolates were collected from four types of clinical specimens from 2015 to 2016 and most of the isolates were from sputum. In terms of clinical characteristics, male and aged patients were more susceptible to an S. maltophilia infection. The majority of included patients had underlying diseases and were hospitalized with associated invasive procedures. The antimicrobial resistance profiles of S. maltophilia isolates showed the highest frequency of resistance to ceftazidime and the lower frequency of resistance to chloramphenicol, levofloxacin, trimethoprim/sulfamethoxazole (TMP/SMX), and no resistance to minocycline. The predominant antibiotic resistance genes among the 200 isolates were the smeF gene (91.5%), followed by blaL1 and blaL2 genes (43% and 10%), respectively. Other antibiotic resistance genes detected were floR (8.5%), intI1 (7%), sul1 (6%), mfsA (4%) and sul2 (2%). Most S. maltophilia isolates could produce biofilm and could swim in a semisolid medium, however, none of the isolates could swarm. All isolates were positive for hemolysin production, whereas 91.5% and 22.5% of isolates could release protease and lipase enzymes, respectively. In MLST analysis, a high degree of genetic diversity was observed among the 200 S. maltophilia isolates. One hundred and forty-one sequence types (STs), including 130 novel STs, were identified and categorized into six different clonal complex groups. The differences in drug resistance patterns and genetic profiles exhibited various phenotypes of biofilm formation, motility, toxin, and enzymes production which support this bacterium in its virulence and pathogenicity. This study reviewed the characteristics of genotypes and phenotypes of S. maltophilia from Thailand which is necessary for the control and prevention of S. maltophilia local spreading.