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Stenotrophomonas maltophiliais a species with immensely broad phenotypic and genotypic diversity that could widely distribute in natural and clinical environments. However, little attention has been paid to reveal their genome plasticity to diverse environments. In the present study, a comparative genomic analysis of S. maltophiliaisolated from clinical and natural sources was systematically explored its genetic diversity of 42 sequenced genomes. The results showed that S. maltophiliaowned an open pan-genome and had strong adaptability to different environments. A total of 1612 core genes were existed with an average of 39.43% of each genome, and the shared core genes might be necessary to maintain the basic characteristics of those S. maltophiliastrains. Based on the results of the phylogenetic tree, the ANI value, and the distribution of accessory genes, genes associated with the fundamental process of those strains from the same habitat were found to be mostly conserved in evolution. Isolates from the same habitat had a high degree of similarity in COG category, and the most significant KEGG pathways were mainly involved in carbohydrate and amino acid metabolism, indicating that genes related to essential processes were mostly conserved in evolution for the clinical and environmental settings. Meanwhile, the number of resistance and efflux pump gene was significantly higher in the clinical setting than that of in the environmental setting. Collectively, this study highlights the evolutionary relationships of S. maltophiliaisolated from clinical and environmental sources, shedding new light on its genomic diversity.