Pythium insidiosum causes a life-threatening infectious disease, called pythiosis, in humans and animals worldwide. Diagnosis of pythiosis is difficult and often delayed. Surgical removal of infected tissue is the main treatment option. Disabilities and death are common outcomes for pythiosis patients. Reports of P. insidiosum infections are rising. While it would be useful for clinical, epidemiological, and microbiological studies, information on genetic variation in P. insidiosum strains is limited. This limitation is, at least in part, due to the cost and time-requirements of DNA sequencing procedures. rDNA-sequence-based phylogenetic analyses categorize P. insidiosum into three groups, in relation to geographic distribution: Clade-I (American strains), Clade-II (American, Asian, and Australian strains), and Clade-III (mostly Thai strains). In rDNA sequence analyses, we observed single nucleotide polymorphisms (SNP) that were associated with the phylogenetic clades of P. insidiosum. In this study, we aim to develop a multiplex PCR assay, targeting the identified SNPs, for rapid genotyping of P. insidiosum. We also aim to assess diagnostic efficiency of the assay for identification of P. insidiosum. Fifty isolates of P. insidiosum and 20 negative-control fungi were recruited for assay evaluation. Based on the pattern of amplicons, the multiplex PCR correctly assigned phylogenetic clades in 98% of the P. insidiosum isolates tested. The assay exhibited 100% sensitivity and specificity for identification of P. insidiosum. In conclusion, the multiplex PCR assay provided accurate, sensitive and specific results for identifying and genotyping P. insidiosum. Thus, this assay could be a simple, rapid, and cost-effective alternative to DNA sequencing for the identification and genotyping of P. insidiosum.