Background Sarcocystis species are obligatorily heteroxenous parasites, of which some are zoonotic, representing a public health and economic impact. This study investigated the occurrence of Sarcocystis spp. in cattle sampled from a Belgian slaughterhouse. Methods A total of 200 carcasses were included in the study, sampled during 10 sampling days. The sedimentation method was applied to isolate the sarcocysts from both heart and diaphragm muscles collected from each carcass. Multiplex PCR, PCR-RFLP as well as cox1 gene sequencing techniques were applied serially on collected sarcocysts for species identification. Results Sarcocystis spp. were detected in 64% (128/200; 95% CI 57-71%) of the sampled carcasses. Female dairy cattle presented the highest Sarcocystis occurrence rate (91%) as well as the highest Sarcocystis species diversity compared to female beef and male beef. Sarcocystis spp. were detected more often in the heart muscles than in the diaphragm among female beef (p < 0.001) and dairy carcasses (p = 0.001), while in male carcasses no significant difference was observed (p = 0.763). The effect of age was not significant in male carcasses (p = 0.872), while the odds of finding sarcocysts significantly increased with age (p = 0.003) within both types of female carcasses. S. cruzi was the most prevalent species and was found in 56.5% (113/200) of the carcasses, followed by S. hominis (21.0%, 42/200), S. bovifelis (12.5%, 25/200), S. bovini (2.0%, 4/200), S. hirsuta (1.5%, 3/200) and S. heydorni (0.5%, 1/200). Six different species were detected in the diaphragm, while only two species were recovered from the heart. S. cruzi was the most prevalent species in heart, while in the diaphragm, this was S. hominis. Conclusions The detection of S. hominis in 21% of the sampled carcasses presents a potential food safety issue, and further research is warranted into controlling this infection. Graphic