The purpose of the present study was to obtain sarcocysts of Sarcocystis buffalonis and Sarcocystis levinei from water buffaloes and characterize the isolates by molecular methods in order to determine whether the two species were genetically different from Sarcocystis hirsuta and Sarcocystis cruzi, respectively, from cattle, which had been characterized before. About 35 macroscopically visible (3-4 × 1-2 mm) and 20 barely visible (1-3 × 0.2 mm) sarcocysts were excised from the esophagus of 18 naturally infected and freshly slaughtered adult water buffaloes at three slaughterhouses in Egypt. Genomic DNA was extracted from the sarcocysts, and all isolates were first characterized at the mitochondrial cytochrome c oxidase subunit I gene (cox1) gene through PCR amplification and direct sequencing. Selected isolates were subsequently further characterized at the 18S and 28S ribosomal (r) RNA genes and the internal transcribed spacer 1 (ITS1) region of the nuclear rDNA unit by direct sequencing or cloning. Only six of the isolated macroscopic sarcocysts belonged to S. buffalonis, whereas the others belonged to Sarcocystis fusiformis. Twelve of the smaller cysts belonged to S. levinei and seven to Sarcocystis sinensis. The characterization of the sarcocysts of S. sinensis and some of the sarcocysts of S. fusiformis have been reported before. Fifteen additional sarcocyst isolates of S. fusiformis were characterized at cox1 in the present study and found to be identical or closely similar to previous isolates. At cox1, the sequence identity between the six isolates of S. buffalonis was 99.8-100 % (two haplotypes), whereas the identity between the 12 isolates of S. levinei was 99.0-100 % (10 haplotypes). The identity between cox1 sequences of S. buffalonis and S. hirsuta (n = 56) was 92.9-93.6 % (on average 93.4 %), and the identity between cox1 sequences of S. levinei and S. cruzi (n = 22) was 92.9-94.0 % (on average 93.5 %). The phylogenetic analyses placed with high support the cox1 sequences of S. buffalonis and S. hirsuta into two monophyletic sister groups, and the same was true for the cox1 sequences of S. levinei and S. cruzi. Hence, the study established that S. buffalonis and S. levinei are distinct species different from S. hirsuta and S. cruzi, respectively. Nucleotide sequences of S. buffalonis could be distinguished from those of S. hirsuta also at the 28S rRNA gene (clearly different) and the ITS1 region (small and uncertain difference) but not at the 18S rRNA gene. Sequences of S. levinei could be distinguished from those of S. cruzi both at the 18S and 28S rRNA genes (ITS1 region not examined). However, the cox1 gene was superior to the 18S and 28S rRNA genes as regards the ability to unambiguously delimit the species within each species pair, since at the latter markers, the number of consistent nucleotide differences between the species was low and there was a slight overlap between the intraspecific and interspecific sequence divergence. Comparison of the newly generated 18S rRNA gene sequences of S. levinei from water buffaloes with similar sequences deposited in GenBank suggested that S. levinei and S. cruzi are not strictly intermediate host specific but might occasionally infect cattle and water buffaloes, respectively.