Sarcocystis spp. are ubiquitous protozoan parasites that can form cysts in striated muscle and CNS of cattle. Cattle hearts are commonly infected by microscopic sarcocysts. Humans can get infected by consuming cattle meat containing the zoonotic parasites Sarcocystis hominis and Sarcocystis heydorni. However, bovine myocardium is generally infected by Sarcocystis cruzi. The aims of this study were to investigate the occurrence of sarcocysts and the identity of Sarcocystis species present in cattle hearts destined to human consumption in the Central region of the Rio Grande do Sul state, southern Brazil. A total of 314 cattle myocardium samples collected from a local abattoir were microscopically examined for the presence of sarcocysts. The sarcocysts isolated from 134 of these samples (ten sarcocysts per sample) were subjected to DNA extraction and PCR amplification. The PCR-amplified DNA fragments were digested with the restriction enzymes BclI and RsaI (PCR-RFLP) for differentiation among S. cruzi, S. hirsuta, and S. hominis. Sarcocystis species identification was confirmed using DNA sequencing of the cox1 mitochondrial DNA. Sarcocysts were detected in all the bovine myocardium samples. PCR-RFLP analysis resulted in successful amplification of 78 of the 134 samples tested. Only the S. cruzi DNA restriction pattern was identified from all of the 78 amplified samples. DNA sequencing also confirmed the presence of S. cruzi DNA. In conclusion, all myocardium samples evaluated were infected with microscopic sarcocysts. S. cruzi was the only species detected infecting the cattle hearts.