Blastocystis is a prevalent infectious agent found in the gastrointestinal tract of humans and animals. While the morphology of Blastocystis has been extensively studied, there is still a lack of comprehensive research on its ultrastructure, especially regarding surface characteristics and their correlation with pathogenic potential. Additionally, the subtyping of Blastocystis does not provide information on the isolate’s pathogenicity. This study aimed to examine the morphology and the cell surface of Blastocystis in avian and non-human primates, including peafowl, pheasant, and lion-headed tamarin. By employing light microscopy and scanning electron microscopy (SEM), this study provides the first evidence of the cellular and surface features of Blastocystis in these animal species. Our findings revealed distinct variations in cell size, shape, and surface morphology among the different host species. Notably, the isolates from peafowl exhibited larger cell sizes compared to the isolates from the pheasant. However, interestingly, both animal species were found to exhibit the same Blastocystis ST6. It was also observed that the surface structure of Blastocystis from different hosts displayed a diverse range of patterns, including mesh-like appearances, deep indentations, and attachments to bacteria. Additionally, findings also revealed the presence of a rough surface structure in peafowl, a characteristic that has been previously linked to pathogenicity and symptomatic infection in animals, as indicated by earlier studies. The findings contribute to our understanding of the morphological features and the surface characteristic of Blastocystis in different host species, shedding light on the parasite’s adaptations and potential implications for host health.