Blastocystis is a ubiquitous waterborne parasite that has been implicated in some disease conditions including colorectal cancer and irritable bowel syndrome, and its surface coat characteristics have been associated with its pathogenicity. Although the morphology of Blastocystis isolates from human and animal sources have been studied, there is a paucity of data on the surface ultrastructure of Blastocystis isolated from water sources. Therefore, this study aimed to determine the occurrence and the ultrastructural surface of Blastocystis isolates from several water sources in Kedah and Penang, Malaysia. A total of 12 water samples were collected, namely, Pinang River and USM Harapan Lake all in Penang, whereas Lata Bayu Waterfall in Baling and UniSHAMS Lake, Kuala Ketil in Kedah. These were examined for Blastocystis by centrifugation and in vitro cultivation. Scanning electron microscopy (SEM) and light microscopy were employed to study the morphological characteristics and the surface ultrastructure of the parasite. Polymerase chain reaction (PCR) was carried out to obtain the subtypes (ST) of the positive Blastocystis isolates. The result revealed 25.0% (3/12) contamination with Blastocystis in which ST1, ST2, and an unknown ST (with a high similarity to ST1) were detected in water samples from the upstream, downstream, and midstream, respectively of Pinang River. Our study also revealed similarities in the sizes of the isolates from different river points, which were notably more diminutive compared to the sizes of the parasites observed in existing data from human and animal isolates. The surface characteristics showed a collection of single and dividing cells with smooth, folded surfaces enclosed in a film-like layer. Additionally, there were roundish, irregularly shaped cells with rough surfaces, and a woolly appearance. This study has added to our knowledge of the surface ultrastructure of Blastocystis and its possible contribution to the pathogenicity of the parasite.