Stenotrophomonas maltophilia is an important nosocomial pathogen. The increased isolates of S. maltophilia among hematology unit patients led to an epidemiological survey. Over 26 months, 24 strains isolated from 23 patients and an environmental isolate from blood pressure armband have been identified. The isolated were first analysed by the use of phenotypical markers: biotype, antibiotic susceptibility, but the minor differences observed justified a genotypic analysis. Pulsed field gel electrophoresis of genomic DNA was carried with XbaI and DraI restriction endonucleases by contour-clamped homogeneous electric field method (CHEF). The data obtained showed a great genomic diversity within the species S. maltophilia. Nevertheless, the same restriction profile was found for 3 patients and 3 other profiles were obtained for 3 couples of patients hospitalized at the same time. All the other strains isolated from subjects hospitalized during the same period exhibited pulsotypes independent from each other. Compared to biotypes and antibiotic susceptibility, these results indicate field only with non modified primers, and the absence of 460 mutations was confirmed by sequencing. Two isolates P1 and P2, from a transplanted patient were amplified with both primers MCMM and MCMW: sequencing analysis shown the presence of a mixture of strains, one of them harbouring A- > G 1378 mutation. One resistant strain was amplified neither with MCMM nor with MCMW: a C- > T silent mutation at nt 1368 was present. As sequencing analysis confirmed PCR results, discriminative PCR enables isolates to be rapidly assessed for the presence or absence of 460 mutations. Moreover, it can distinguish Met to Val from Met to Ile mutations, and allows the analysis of mixtures of sensitive and resistant strains.