Burkholderia pseudomallei, a Gram-negative bacterial pathogen that causes melioidosis, is of public health importance in endemic areas including Malaysia. An investigation of the molecular epidemiology links of B. pseudomallei would contribute to better understanding of the clonal relationships, transmission dynamics and evolutionary change. Multi-locus sequence typing (MLST) of 45 clinical B. pseudomallei isolates collected from sporadic meliodosis cases in Malaysia was performed. In addition, a total of 449 B. pseudomallei Malaysian strains submitted to the MLST database from 1964 until 2019 were included in the temporal analysis to determine the endemic sequence types (STs), emergence and re-emergence of ST(s). In addition, strain-specific distribution was evaluated using BURST tool. Genotyping of 45 clinical strains were resolved into 12 STs and the majority were affiliated with ST46 (n=11) and ST1342 (n=7). Concomitantly, ST46 was the most prevalent ST in Malaysia which first reported in 1964. All the Malaysian B. pseudomallei strains were resolved into 76 different STs with 36 of them uniquely present only in Malaysia. ST1342 was most closely related to ST1034, in which both STs were unique to Malaysia and first isolated from soil samples in Pahang, a state in Malaysia. The present study revealed a high diversity of B. pseudomallei in Malaysia. Localised evolution giving rise to the emergence of new STs was observed, suggesting that host and environmental factors play a crucial role in the evolutionary changes of B. pseudomallei.