Melioidosis is an infection that occurs in humans and a wide range of animals by Burkholderia pseudomallei as a causative pathogen, which inhabits environments of tropical and subtropical climate zones, particularly in Thailand. The standard laboratory diagnosis is by culture and biochemical characteristic differentiation. This study aimed to determine the diversity of colony morphology, biochemical characteristics, and drug susceptibility patterns of a B. pseudomallei collection isolated from humans, animals, soil, and water in Thailand. According to the previous characterization of colony morphotypes from Type I to VII, Type I is predominant with B. pseudomallei from humans and animals with a similar water source. Meanwhile, Type III is predominant from soil sources. The results confirmed the hypothesis that infection might be from water instead of soil exposure. The typical biochemical phenotypes were consistent among different isolate sources, including cytochrome oxidase (positive), triple iron sugar (K/N or A/N), sulfide (negative), indole (negative), non-motile, glucose oxidizing, and DNase (positive). The biochemical characteristic results supported the data for the selection of the prepopulated biochemical tests in clinical and research laboratories. The antimicrobial susceptibility patterns by standard disk diffusion showed mode values of inhibition zones for selected drugs that had lower trends in B. pseudomallei isolated from the soil than other sources. The results were consistent with MIC50 and MIC90 values of ceftazidime, which was highest in B. pseudomallei from the soil than other sources. © 2022, Walailak University. All rights reserved.