To provide further evidence of waterborne transmission of Blastocystis, a total of 31 wastewater treatment plants from geographically distinct locations across the Philippines were sampled for influent and effluent sewage samples. In vitro cultivation was the method of choice to increase sensitivity of detection. Blastocystis cysts were detected in 15% (9/62) of the samples using in vitro culture. Moreover, influent and effluent samples were 23% (7/31) and 7% (2/31) positive for the parasite, respectively. The presence of viable cysts in treated samples may be an indication of the inefficiency of the treatment process in preventing Blastocystis from entering the environment. Polymerase chain reaction and sequencing of the full-length small subunit ribosomal RNA (SSU rRNA) genes of the nine wastewater isolates were performed. The SSU rRNA gene sequences of the isolates showed very high similarity (98 to 99%) to homologous sequences of Blastocystis described previously. The phylogenetic tree constructed showed that the wastewater isolates clustered with each other with good bootstrap support and belonged to two subtypes (ST) - ST1 and ST2. This is the first report of subtyping Blastocystis isolates from wastewater samples and gives further emphasis to the remarkable genetic diversity of the parasite.