Sarcocystis spp. are protozoan parasites with an obligatory two-host life cycle, with herbivores as intermediate hosts and carnivores as definitive hosts. Cattle are intermediate hosts for several species of Sarcocystis: indeed, in addition to S. cruzi, S. hirsuta and S. hominis, at least four new species were recently identified in bovine muscle: S. bovifelis, S. rommeli, S. bovini and S. heydorni. Since is not possible to unambiguously discriminate between S. hominis and the new species either morphologically or by the analysis of the 18S ribosomial (rRNA) gene, the aim of the present study was to use molecular techniques to discriminate cattle Sarcocystis species, taking advantage of the higher discriminative power of the Cytochrome C Oxidase subunit I mitochondrial (mtDNA COI) gene. Therefore, 119 bovine muscle samples were tested to identify S. hominis-like sarcocystis using a multiplex PCR of the 18S rRNA gene; later, positive samples were tested using a newly designed primer set for the PCR amplification of COI gene. Species identification was achieved by sequencing the amplified products: 16 sequences were confirmed to belong to S. bovifelis, while 12 sequences didn’t constitute the best BLAST match of any of the published sequences, allowing to speculate the possible presence of S. hominis. This study confirms the higher discriminatory power of COI mitochondrial gene; besides, our work provides the first report of S. bovifelis in Italy.