Objective: To determine the genetic diversity of Plasmodium (P.) knowlesi isolates from Sabah, Malaysian Borneo and Peninsular Malaysia, targeting the S-type SSU rRNA gene and including aspects of natural selection and haplotype. Methods: Thirty-nine blood samples infected with P. knowlesi were collected in Sabah, Malaysian Borneo and Peninsular Malaysia. The S-type SSU rRNA gene was amplified using polymerase chain reaction, cloned into a vector, and sequenced. The natural selection and haplotype of the S-type SSU rRNA gene sequences were determined using DnaSP v6 and illustrated using NETWORK v10. This study's 39 S-type SSU rRNA sequences and eight sequences from the Genbank database were subjected to phylogenetic analysis using MEGA 11. Results: Overall, the phylogenetic analysis showed no evidence of a geographical cluster of P. knowlesi isolates from different areas in Malaysia based on the S-type SSU rRNA gene sequences. The S-type SSU rRNA gene sequences were relatively conserved and with a purifying effect. Haplotype sharing of the S-type SSU rRNA gene was observed between the P. knowlesi isolates in Sabah, Malaysian Borneo, but not between Sabah, Malaysian Borneo and Peninsular Malaysia. Conclusions: This study suggests that the S-type SSU rRNA gene of P. knowlesi isolates in Sabah, Malaysian Borneo, and Peninsular Malaysia has fewer polymorphic sites, representing the conservation of the gene. These features make the S-type SSU rRNA gene suitable for comparative studies, such as determining the evolutionary relationships and common ancestry among P. knowlesi species.