Forty-three Pythium insidiosum clinical isolates recovered from human pythiosis cases in Thailand were characterized by random amplified polymorphic DNA (RAPD) analysis. Three random oligonucleotide primers, OPW11, OPW12 and OPX13 generated 39, 34 and 35 DNA patterns with high value of typeability (100%), reproducibility (98.5, 88.8 and 93.3%) and discriminatory power (0.83, 0.82 and 0.77), respectively. Using GelCompar software based on band similarity, the 43 clinical isolates of P. insidiosum could be arranged into 9, 13 and 11 clades using OPW11, OPW12 and OPX13, respectively and the combination of all three primers revealed 36 RAPD patterns. Members in each RAPD pattern varied in both clinical forms and/or geographical locations. RAPD pattern 15 was found in 6 isolates, half of which were found in central region of Thailand. Isolates MCC15 and MCC16 isolated from different patients exhibited identical pattern with all three primers. Our results revealed high genetic heterogeneity among Pythium insidiosum isolates in Thailand. RAPD method should be appropriate for future epidemiological studies of P. insidiosum strains from patients and from natural habitats.